SHH Medulloblastoma Research Articles

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.

OS09.1.AIN VIVOTWO-PHOTON TUMOR IMAGING IN AN ORTHOTOPIC MEDULLOBLASTOMA MOUSE MODEL

Abstract BACKGROUND Medulloblastoma represents the most common primary brain malignancy in pediatric patients and is associated with neuro-cognitive impairment and frequent relapses after aggressive multimodal therapy. Adoptive immunotherapies such as CAR T-cell therapy are currently being investigated, showing promising results in preclinical models but lacking efficacy in first-in-human trials. Refined in vivo models to investigate reasons for treatment failures are therefore urgently needed. METHODS We developed a xenogeneic orthotopic medulloblastoma (MB) model in mice by combining a chronic cerebellar window with repetitive intravital two-photon laser scanning microscopy. Red tdTomato-fluorescent DAOY (SHH MB) tumor cells expressing EphA2 were implanted intracranially into the cerebellum of immunodeficient mice (n = 7), and tumor cell formation was followed by in vivo-microscopy. Intravenous fluorescein isothiocyanate (FITC)-dextran was used for intravascular plasma staining. EphA-directed CAR T-cells were injected into the adjacent brain parenchyma. RESULTS Chronic cranial window implantation was well tolerated and allowed repetitive visualization of the mouse cerebellum. After intracranial tumor cell implantation, continuous tumor growth could be evaluated with epifluorescence as well as 2-photon laser scanning microscopy. Tumor formation was identified as early as day 5 in every mouse and could continuously be followed up to 35 days until mice succumbed due to tumor burden. Window quality and fluorescence intensity remained high until the end of the experiments. Studies on GFP-expressing CAR T-cell injection directed against EphA2 are currently ongoing. CONCLUSIONS We herein establish a robust orthotopic medulloblastoma mouse model that allows repetitive in vivo tracking of fluorescence-expressing tumor cells, CAR T-cells and blood vessels over weeks. Such models may be used to study medulloblastoma tumor growth under the influence of different immunotherapies and reveal interactions between tumor cells and CAR T-cell therapies on a single-cell level.

Medulloblastoma

In the 5th edition of the WHO classification, medulloblastomas, which are representative pediatric brain tumors, are categorized into four groups: WNT, SHH-TP53 wild, SHH-TP53 mutant, and non-WNT/non-SHH, based on their molecular background. While the histopathological findings still hold importance in predicting prognosis, the histopathological classification is no longer utilized in this edition. SHH medulloblastomas are further subdivided into two groups based on the presence or absence of TP53 mutation, as their clinical characteristics and prognosis differ. Group 3 and Group 4 medulloblastomas, recognized as distinct molecular groups in clinical practice, are combined into a single group called "non-WNT/non-SHH", because they lack specific molecular pathway activation. Furthermore, based on methylation profiling, dividing SHH medulloblastoma into four subgroups and non-WNT/non-SHH medulloblastoma into eight subgroups was proposed. Understanding the unique clinical characteristics and prognosis associated with each group is crucial. However, it is important to acknowledge that our current understanding of prognosis is based on treatment approaches guided by clinical risk factors such as postoperative residual tumor volume and the presence of metastatic disease. This molecular-based classification holds promise in guiding the development of optimal treatment strategies for patients with medulloblastoma.

SHH medulloblastoma and very early onset of bowel polyps in a child with PTEN hamartoma tumor syndrome.

Phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome (PHTS) is a cancer predisposition syndrome characterized by an increased risk of developing benign and malignant tumors, caused by germline pathogenic variants of the PTEN tumour suppressor gene. PTEN gene variants often present in childhood with macrocephaly, developmental delay, and/or autism spectrum disorder while tumors and intestinal polyps are commonly detected in adults. PHTS is rarely associated with childhood brain tumors with only two reported cases of medulloblastoma (MB). We report the exceptional case of an infant carrying a germline and somatic pathogenic variant of PTEN and a germline and somatic pathogenic variant of CHEK2 who developed a MB SHH in addition to intestinal polyposis.

OTHR-13. IDENTIFYING INDIVIDUALS WITH PRIMARY CENTRAL NERVOUS SYSTEM TUMORS AT RISK FOR HEREDITARY CANCER SYNDROMES USING A STATEWIDE CANCER REGISTRY

Abstract INTRODUCTION Central Nervous System (CNS) tumors are associated with hereditary cancer syndromes (HCS), especially among pediatric and AYA populations. Using population-based resources, we evaluated the proportion of patients meeting HCS testing criteria. METHODS Cases of CNS tumors diagnosed in individuals ages 0-39 were identified through the Utah Cancer Registry. Using the Utah Population Database, cases were linked to cancer family history and, when available, to HCS genetic data. A genetic counselor and pediatric neuro-oncologist reviewed family pedigrees containing ≥2 CNS tumors. RESULTS We identified 347 CNS tumors in 320 individuals, from 161 families (FSIR P&amp;lt;0.05). The most common tumors were: astrocytoma (n=93), pituitary (n=38), glioma (n=34, 4 optic nerve), glioblastoma (n=32), meningioma (n=31), oligodendroglioma (n=24), and medulloblastoma (n=20). Sixteen individuals had multiple cancers: 9 had ≥ 2 CNS tumors, 7 had CNS and non-CNS tumors. Of those with multiple tumors, 3 met criteria for NF2, 1 for NF1, and 1 for Chompret. 213 individuals (67%) met criteria for HCS testing based on their personal cancer history. Criteria met included glial tumor (&amp;lt; age 40) (n=163), NF2 (n=14), NF1 (n=12), rhabdoid tumor predisposition (n=10), Chompret (n=7), VHL (n=3), medulloblastoma—SHH (n=3), DICER1 (n=1). Germline data were available for 23 individuals, and 14 had a germline mutation (Li Fraumeni Syndrome=8, VHL=3, APC=1, DICER1=1). An additional 12 people met Chompret criteria based on their CNS tumor plus family history. Of these 4 had germline testing, of which 3 had LFS. CONCLUSIONS In our cohort, 70% of individuals met criteria for HCS testing. However, this may be an underestimate because many syndromes associated with CNS tumors also predispose to non-CNS tumors, which were not included in the initial query. Research on the actual prevalence of HCS in individuals with CNS tumors requires germline data in a queryable form in the medical record.

IMMU-14. WAKING THE SLEEPING GIANT: TLR7/8 AGONIST TREATMENT STIMULATES TUMOR-ASSOCIATED MYELOID CELLS FOR MEDULLOBLASTOMA IMMUNOTHERAPY

Abstract Medulloblastomas contain large myeloid populations and we have identified a novel therapy that effectively recruits these non-tumor cells for anti-cancer effect. Medulloblastoma myeloid cells have bivalent functions, supporting tumor growth through IGF1 secretion and immunosuppression, or inhibiting tumor growth through diverse mechanisms including phagocytosis. We investigated a nanoparticle formulation of the TLR7/8 agonist Resiquimod (POx-Resiquimod), administered to Gfap-Cre/SmoM2 (G-Smo) mice that are engineered to develop SHH medulloblastomas, using animal survival time studies, flow cytometry and single-cell trancriptomics (scRNA-seq). POx-Resiquimod treatment increased the survival of G-Smo mice as a single agent and sensitized the typically radiation-resistant G-Smo tumors to radiation therapy. Mechanistically, POx-resiquimod increased the proportions of myeloid cells in medulloblastoma, while blocking IGF1 secretion and re-polarizing the myeloid population from anti-inflammatory to pro-inflammatory. POx-resiquimod induced phagocytosis-related genes, including CD72 and the scavenger receptor MARCO. Functional studies confirmed that POx-resiquidmod increased tumor cell phagocytosis, linking molecular changes to changes in myeloid cell behavior. Our data show that the innate immune function of myeloid cells can be harnessed for medulloblastoma treatment and identify the nanoparticle-delivered TLR7/8 agonist POx-Resiquimod as a specific and effective new agent for medulloblastoma immunotherapy that can be combined with existing modalities.

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.

Abstract 1708: Sex-biased function of an oncogene, YAP1, regulates medulloblastoma stem cells and immune evasion

Abstract Sex disparity in both cancer incidence and response to therapeutics, especially immunotherapy, has become an important area of research in recent years. Brain tumors show obvious sex biases in incidence and therapy response. Medulloblastoma (MB) is the most common malignant pediatric brain tumor, and similar to other brain tumors, males have consistently higher incidence across all subgroups and at all ages. Moreover, for patients &amp;gt;3 years of age, females have significantly better outcomes than males. However, molecular drivers of such sex disparity in incidence and prognosis remain unclear.Here, we report a male-biased function of an oncogene, YAP1, in MB formation and immune suppression. Integrated multi-platform genomic, transcriptomic, proteomic, and in vivo transcriptional target analyses show that YAP1 promotes cancer stem cell maintenance through concurrent transcriptional activation of stemness genes, such as Sox2, and repression of differentiation regulators such as NeuroD1 and Zic1/2. YAP1 expression in MB cells also inhibits anti-tumor immune cell infiltration by directly regulating Csf1, Igf1, and Igfbp3 transcription and modulating IL6-JAK-STAT3, TNFR1, TGFb, and CCL5 Pathways. Notably, we found that YAP1 genetic deletion results in disproportionately higher survival of males than females in a spontaneous mouse model of infantile SHH MB, despite equivocal expression levels in both sexes. Consistent with our findings in mouse models, YAP1 downstream genes stratify MB patient survival in males but not females. Finally, we show that a brain-penetrant YAP1 inhibitor, verteporfin, extends survival and enhances immune infiltrates in vivo, mimicking the effects of Yap1 genetic deletion. In summary, this study demonstrates that an oncogene function may be sex-biased and underscores the need to incorporate sex as a variable in cancer mechanisms, tumor immunology, and treatment response studies. Citation Format: Nourhan Abdelfattah, Sivaraman Natarajan, Jose Maldonado, Han Nhat Tran, Hannah Borland, Rachael McMinimy, Shu-Hsia Chen, Fernando Camargo, James Olson, Joshy George, Kyuson Yun. Sex-biased function of an oncogene, YAP1, regulates medulloblastoma stem cells and immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1708.

Sex differences in methylation profiles are apparent in medulloblastoma, particularly among SHH tumors.

Medulloblastoma, the most common malignant pediatric brain tumor, displays marked sex differences in prevalence of the four main molecular subgroups: SHH, WNT, Group 3 and Group 4. Males are more frequently diagnosed with SHH, Group 3 and 4 tumors, which have worse prognoses than WNT tumors. Little is known about sex differences in methylation profiles within subgroups. Using publicly available methylation data (Illumina HumanMethylation450K array), we compared beta values for males versus females. Differentially methylated positions (DMP) by sex within medulloblastoma subgroups were identified on the autosomes. DMPs were mapped to genes and Reactome pathway analysis was run by subgroup. Kaplan-Meier survival curves (Log-Rank p-values) were assessed for each sex within subgroup. MethylCIBERSORT was used to investigate the tumor microenvironment using deconvolution to estimate the abundances of immune cell types using DNA methylation data. There were statistically significant differences in sex by medulloblastoma subgroups (chi-squared p-value=0.00004): Group 3 (n=144; 65% male), Group 4 (n=326; 67% male), SHH (n=223; 57% male) and WNT (n=70; 41% male). Females had worse survival than males for SHH (p-value=0.02). DMPs by sex were identified within subgroups: SHH (n=131), Group 4 (n=29), Group 3 (n=19), and WNT (n=16) and validated in an independent dataset. Unsupervised hierarchical clustering showed that sex-DMPs in SHH did not correlate with other tumor attributes. Ten genes with sex DMPs (RFTN1, C1orf103, FKBP1B, COL25A1, NPDC1, B3GNT1, FOXN3, RNASEH2C, TLE1, and PHF17) were shared across subgroups. Significant pathways (p<0.05) associated with DMPs were identified for SHH (n=22) and Group 4 (n=4) and included signaling pathways for RET proto-oncogene, advanced glycosylation end product receptor, regulation of KIT, neurotrophic receptors, NOTCH, and TGF-β. In SHH, we identified DMPs in four genes (CDK6, COL25A1, MMP16, PRIM2) that encode proteins which are the target of therapies in clinical trials for other cancers. There were few sex differences in immune cell composition within tumor subgroups. There are sexually dimorphic methylation profiles for SHH medulloblastoma where survival differences were observed. Sex-specific therapies in medulloblastoma may impact outcomes.

PRC2 disruption in cerebellar progenitors produces cerebellar hypoplasia and aberrant myoid differentiation without blocking medulloblastoma growth

We show that Polycomb Repressive Complex-2 (PRC2) components EED and EZH2 maintain neural identity in cerebellar granule neuron progenitors (CGNPs) and SHH-driven medulloblastoma, a cancer of CGNPs. Proliferating CGNPs and medulloblastoma cells inherit neural fate commitment through epigenetic mechanisms. The PRC2 is an epigenetic regulator that has been proposed as a therapeutic target in medulloblastoma. To define PRC2 function in cerebellar development and medulloblastoma, we conditionally deleted PRC2 components Eed or Ezh2 in CGNPs and analyzed medulloblastomas induced in Eed-deleted and Ezh2-deleted CGNPs by expressing SmoM2, an oncogenic allele of Smo. Eed deletion destabilized the PRC2, depleting EED and EZH2 proteins, while Ezh2 deletion did not deplete EED. Eed-deleted cerebella were hypoplastic, with reduced proliferation, increased apoptosis, and inappropriate muscle-like differentiation. Ezh2-deleted cerebella showed similar, milder phenotypes, with fewer muscle-like cells and without reduced growth. Eed-deleted and Ezh2-deleted medulloblastomas both demonstrated myoid differentiation and progressed more rapidly than PRC2-intact controls. The PRC2 thus maintains neural commitment in CGNPs and medulloblastoma, but is not required for SHH medulloblastoma progression. Our data define a role for the PRC2 in preventing inappropriate, non-neural fates during postnatal neurogenesis, and caution that targeting the PRC2 in SHH medulloblastoma may not produce durable therapeutic effects.

Identifying new biomarkers of aggressive Group 3 and SHH medulloblastoma using 3D hydrogel models, single cell RNA sequencing and 3D OrbiSIMS imaging

The most common malignant brain tumour in children, medulloblastoma (MB), is subdivided into four clinically relevant molecular subgroups, although targeted therapy options informed by understanding of different cellular features are lacking. Here, by comparing the most aggressive subgroup (Group 3) with the intermediate (SHH) subgroup, we identify crucial differences in tumour heterogeneity, including unique metabolism-driven subpopulations in Group 3 and matrix-producing subpopulations in SHH. To analyse tumour heterogeneity, we profiled individual tumour nodules at the cellular level in 3D MB hydrogel models, which recapitulate subgroup specific phenotypes, by single cell RNA sequencing (scRNAseq) and 3D OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) imaging. In addition to identifying known metabolites characteristic of MB, we observed intra- and internodular heterogeneity and identified subgroup-specific tumour subpopulations. We showed that extracellular matrix factors and adhesion pathways defined unique SHH subpopulations, and made up a distinct shell-like structure of sulphur-containing species, comprising a combination of small leucine-rich proteoglycans (SLRPs) including the collagen organiser lumican. In contrast, the Group 3 tumour model was characterized by multiple subpopulations with greatly enhanced oxidative phosphorylation and tricarboxylic acid (TCA) cycle activity. Extensive TCA cycle metabolite measurements revealed very high levels of succinate and fumarate with malate levels almost undetectable particularly in Group 3 tumour models. In patients, high fumarate levels (NMR spectroscopy) alongside activated stress response pathways and high Nuclear Factor Erythroid 2-Related Factor 2 (NRF2; gene expression analyses) were associated with poorer survival. Based on these findings we predicted and confirmed that NRF2 inhibition increased sensitivity to vincristine in a long-term 3D drug treatment assay of Group 3 MB. Thus, by combining scRNAseq and 3D OrbiSIMS in a relevant model system we were able to define MB subgroup heterogeneity at the single cell level and elucidate new druggable biomarkers for aggressive Group 3 and low-risk SHH MB.

GEN-8 COMPREHENSIVE WHOLE GENOME SEQUENCING ANALYSIS ELUCIDATES STRUCTURAL VARIANTS IN MEDULLOBLASTOMA

Abstract Introduction Medulloblastoma is the most common malignant pediatric brain tumor with a poor prognosis. International collaborative efforts for sequencing the genomes of medulloblastomas have delineated the landscape of coding mutations. However, little is yet known about structural variants (SVs). Here, we analyzed whole-genome sequencing (WGS) data of medulloblastoma to reveal the comprehensive genetic aberrations, including SVs. Methods We collected and analyzed publically available WGS data of 432 cases with medulloblastoma from MAGIC consortium, ICGC, and St Jude cloud. Results The median coverage is 36.4x with no difference among cohorts. The median number of SV per case is 1.0 (WNT), 5.0 (SHH), 6.0 (Group 3), and 5.0 (Group 4), respectively. SHH medulloblastoma with TP53 mutation has significantly a greater number of SVs (77.0/sample), suggesting that genome instability by TP53 mutations gives rise to SVs. At least one SV involved in already known driver genes in 10.9% of the SHH cases, supporting a model where SV could lead to tumor initiation. Since some of those SVs are caused by copy-neutral translocation, detecting those alterations is challenging by other than WGS. Complex SVs involving TERT focal amplification are identified in 11.5% of the SHHα subtype which usually does not have TERT promoter mutation. Although TERT promoter mutation is enriched predominantly in the SHHδ subtype, overexpression of TERT may be necessary for tumor maintenance in some of the SHHα cases. Unbalanced amplifications are commonly observed in known drive genes such as PPM1D, CCND2, and PVT1 where a part of exons are exclusively amplified, implying that functionally important regions are selectively altered by SVs which could promote tumor development. Conclusions SV affects not just copy number changes but also the structure of the genome, where breakpoints exist based on gene function and regulations. SVs along with genetic mutations contribute to medulloblastoma pathogenesis.

Targeting H3K27me3 demethylase to inhibit Shh signaling and cholesterol metabolism in medulloblastoma growth.

Previously we uncovered the epigenetic regulation of medulloblastoma that low levels of H3K27me3 are required for Shh target gene expression and medulloblastoma growth. Since Jmjd3, an H3K27me3 demethylase, is responsible for maintaining low H3K27me3 at Shh target genes, targeting Jmjd3 could be an efficient way to inhibit Shh signaling and medulloblastoma growth. Here we show that the small molecule GSK-J4, an inhibitor of Jmjd3, significantly inhibited the expression of Shh target genes in Shh responsive cell models and primary cerebellar granule neuron precursors. GSK-J4 also significantly reduced the growth of primary Shh medulloblastoma cultures. Treating human medulloblastoma cell line DaoY by GSK-J4 led to cell cycle arrest at G0/G1 phase with decreased cells in S-phase. Tumor cell proliferation was significantly inhibited by GSK-J4 treatment. Gene expression analyses showed that GSK-J4 additionally constrained the expression of key genes in cholesterol biosynthesis. Our results highlight the possibility that targeting H3K27me3 demethylase Jmjd3 with GSK-J4 to inhibit Shh signaling and cholesterol metabolism is a potential application to treat Shh medulloblastoma.

IMMU-09. IMMUNOLOGICAL CHARACTERIZATION OF PEDIATRIC BRAIN TUMORS HAS CLINICAL IMPLICATIONS FOR PATIENT MANAGEMENT AND PROGNOSIS

Abstract The tumor immune microenvironment (TIME) is a growing area of interest, however, the extent of immune activation in childhood CNS tumors is unknown. Although immunotherapy has not gained success in most CNS cancers, our group described gliomas with MMRD which exhibit hypermutation and favorable responses to immune checkpoint inhibition (ICI). Therefore, detailed characterization of the CNS TIME is key for the development of novel immunotherapeutic strategies and application of existing ones in childhood brain tumors. We developed a clinical NanoString immune-oncology panel that includes markers reflecting cell types, therapeutic targets, and cellular pathways, as well as the 18-gene tumor inflammation signature (TIS), a biomarker for ICI response. We tested over 500 brain tumors, including 266 low-grade gliomas (LGG), 170 high-grade gliomas (HGG), 91 MMRD tumors, 16 ependymomas, 46 medulloblastomas, and 36 non-tumor brain samples. Overall, ependymomas and medulloblastomas had low levels of inflammation, although SHH medulloblastomas had higher inflammation than other subtypes. IDH-mutant LGG were immunologically cold, while many gliomas with pediatric-LGG mutations had high levels of inflammation, including upregulation of immune checkpoints – indicating that ICI may be an effective strategy. Interestingly, in pediatric-LGG inflammation impacted outcome in tumors with the same genetic alterations. BRAF V600E-mutant LGG exhibiting high TIS had inferior prognosis (p = 0.02), while no such relationship was observed in BRAF-fused tumors. Diffuse midline gliomas had higher inflammation than hemispheric HGG, indicating that these tumors are not immunologically cold, as has been previously reported. In MMRD tumors treated with ICI, high TIS correlated with improved survival and was independent from hypermutation and mutational burden. Furthermore, MMRD gliomas had high expression of several other immune checkpoints including LAG3, suggesting its value as an additional therapeutic target. In summary, characterization of the TIME across pediatric brain tumors provides potential prognostic clues and suggest treatment strategies for further investigation.

PATH-31. ECDNA PROMOTES TUMORIGENICITY AND INTRATUMORAL HETEROGENEITY IN MEDULLOBLASTOMA

Abstract Extrachromosomal circular DNA (ecDNA) is an important driver of aggressive cancers, including medulloblastoma (MB), the most common pediatric brain tumor. To assess the clinical importance of ecDNA in MB, we applied computational methods to detect ecDNA in the genomes of a cohort of 468 MB patients and 31 MB model systems. Among patients, ecDNA was detected in 18% of tumors and carried over twofold greater risk of mortality. Affected genomic loci harbor up to hundredfold amplification of oncogenes including MYC, MYCN, TERT, and other novel putative oncogenes. Between sequential patient biopsies at initial diagnosis and subsequent relapse, we observed structural variation at ecDNA loci and generation of new ecDNA sequences. Among model systems, ecDNA was found in 19 of 31 genomes (61%). Although ecDNA was far more prevalent among MB models than patients, the ecDNA genomic sequences were conserved between most patient-derived xenograft (PDX) models and the human tumors from which they were made. To elucidate the functional regulatory landscapes of ecDNAs in MB, we generated transcriptional (RNA-seq), accessible chromatin (ATAC-seq), and chromatin interaction (Hi-C) profiles of 6 MB tumor samples. In each case, we identified regulatory interactions that cross fusion breakpoints on the ecDNA, representing potential “enhancer rewiring” events which may contribute to transcriptional activation of co-amplified oncogenes. To test this hypothesis, we conducted an in vitro CRISPRi screen targeting regulatory regions on the ecDNA of a MB cell line and identified distal enhancers which promote proliferation. Using single-cell sequencing, we have also developed methods to explore intratumoral heterogeneity of ecDNA in a p53-mutant SHH MB patient tumor and its corresponding PDX model. In summary, our study analyzes the frequency, diversity, and functional relevance of ecDNA across MB subgroups and provides strong justification for continued mechanistic studies of ecDNA in MB with the potential to uncover new therapeutic approaches.

TMET-01. DISRUPTION OF THE SERINE-PRODUCING PATHWAYS SLOWS PROGRESSION OF THE SONIC HEDGEHOG-DRIVEN MEDULLOBLASTOMA

Abstract We have found that medulloblastoma metabolism is specialized to promote growth in the CNS and can be targeted for anti-tumor therapy. Prior studies show that serine is scarce in the CNS and that metastatic tumors require serine-producing enzymes to grow in the brain. We analyzed whether medulloblastoma, a primary brain tumor, showed similar serine dependency. We found that Sonic Hedgehog signaling, which induces proliferation in cerebellar granule neuron progenitors in normal development and medulloblastoma formation when hyperactivated, also induces PHGDH and SHMT1, which each catalyze key steps in different serine-production pathways. To define the functional role of PHGDH and SHMT1 in medulloblastomas, we bred mice genetically engineered to develop SHH medulloblastomas with mice carrying deletions of each of these genes. We found that mice with Phgdh-deleted medulloblastomas showed slower progression and longer survival times, compared to mice with Phgdh-intact medulloblastomas. Medulloblastomas with combined deletion of Phgdh and Shmt1 show even slower tumor progression. Stable-isotope flux analysis suggests that different serine-producing pathways compensate for the loss of individual serine-producing enzymes, providing a rationale for the greater anti-tumor effect of disrupting multiple serine-producing enzymes simultaneously. Our data show that serine-producing enzymes can be targeted as a novel approach to medulloblastoma therapy, and underscore the importance of targeting multiple serine-production pathways in complex metabolic interventions.

Clinical and Molecular Features in Medulloblastomas Subtypes in Children in a Cohort in Taiwan.

Medulloblastoma (MB) was classified into four molecular subgroups: WNT, SHH, group 3, and group 4. In 2017, 12 subtypes within 4 subgroups and 8 subtypes within non-WNT/non-SHH subgroups according to the differences of clinical features and biology were announced. In this study, we aimed to identify the heterogeneity of molecular features for discovering subtype specific factors linked to diagnosis and prognosis. We retrieved 70 MBs in children to perform RNA sequencing and a DNA methylation array in Taiwan. Integrated with clinical annotations, we achieved classification of 12 subtypes of pediatric MBs in our cohort series with reference to the other reported series. We analyzed the correlation of cell type enrichment in SHH MBs and found that M2 macrophages were enriched in SHH β, which related to good outcomes of SHH MBs. The high infiltration of M2 macrophages may be an indicator of a favorable prognosis and therapeutic target for SHH MBs. Furthermore, C11orf95-RELA fusion was observed to be associated with recurrence and a poor prognosis. These results will contribute to the establishment of a molecular diagnosis linked to prognostic indicators of relevance and help to promote molecular-based risk stratified treatment for MBs in children.

Chromosomal Instability Characterizes Pediatric Medulloblastoma but Is Not Tolerated in the Developing Cerebellum.

Medulloblastoma is a pediatric brain malignancy that consists of four transcriptional subgroups. Structural and numerical aneuploidy are common in all subgroups, although they are particularly profound in Group 3 and Group 4 medulloblastoma and in a subtype of SHH medulloblastoma termed SHHα. This suggests that chromosomal instability (CIN), the process leading to aneuploidy, is an important player in medulloblastoma pathophysiology. However, it is not known if there is ongoing CIN in medulloblastoma or if CIN affects the developing cerebellum and promotes tumor formation. To investigate this, we performed karyotyping of single medulloblastoma cells and demonstrated the presence of distinct tumor cell clones harboring unique copy number alterations, which is suggestive of ongoing CIN. We also found enrichment for processes related to DNA replication, repair, and mitosis in both SHH medulloblastoma and in the highly proliferative compartment of the presumed tumor cell lineage-of-origin, the latter also being sensitive to genotoxic stress. However, when challenging these tumor cells-of-origin with genetic lesions inducing CIN using transgenic mouse modeling, we found no evidence for large chromosomal aberrations in the cerebellum or for medulloblastoma formation. We therefore conclude that without a background of specific genetic mutations, CIN is not tolerated in the developing cerebellum in vivo and, thus, by itself is not sufficient to initiate medulloblastoma.

Medulloblastoma and Down Syndrome: An Extremely Rare Association.

Medulloblastoma has a reduced incidence in Down syndrome (DS). This protective characteristic has not been clarified yet. Here, we report the second case of SHH medulloblastoma and DS documented in the literature. A complete surgery was performed followed by reduced craniospinal irradiation dose and adjuvant chemotherapy. No evidence of tumor recurrence was observed. The overall survival was 9.1 years. No family history or physical stigma of other hereditary predisposition syndrome was found. In the elucidation of this extremely rare association, future case reports play an important role in defining the spectrum of brain tumors and their peculiar features in DS.

Abstract 6214: Detecting and characterizing extrachromosomal DNA in medulloblastoma at single-cell resolution

Abstract Extrachromosomal circular DNA (ecDNA) is an important driver of aggressive cancers, including medulloblastoma (MB), the most common malignant pediatric brain tumor. To assess the clinical import of ecDNA in MB, we applied computational methods to detect ecDNA to the genomes of a cohort of 468 MB patients and 31 MB model systems. Among patients, ecDNA was detected in 18% of tumors and carried threefold greater risk of mortality. Between sequential patient biopsies at initial diagnosis and subsequent relapse, we observed frequent structural variation at ecDNA loci and generation of new ecDNA sequences. Among model systems, ecDNA was found in 19 of 31 genomes (61%). Although ecDNA was far more prevalent among MB models than patients, the ecDNA genomic sequence was conserved between most patient-derived xenograft (PDX) models and their origin human tumors. EcDNA promotes high oncogene copy number, accelerated evolution of drug resistance and poor prognosis. Mechanistically, these behaviors are hypothesized to be consequences of intratumoral heterogeneity of ecDNA copy number; however, there are currently no methods to quantify the abundance and distribution of ecDNA across single cells in a tumor. We have previously identified a primary TP53-mutant SHH medulloblastoma tumor, which harbors multiple distinct lineages of ecDNA originating from different chromosomes. We have now simultaneously analyzed the accessible chromatin and gene expression in single cells of the human tumor and of a orthotopic mouse xenograft (PDX) derived for this tumor, using multiome single-cell ATAC-seq and gene expression (10X Genomics). By measuring scATAC-seq read depth at the ecDNA loci relative to the rest of the genome, we estimate ecDNA copy number in single cells. We observe substantial variation in the ecDNA copy number of cells in both the human tumor and the PDX, and strong association with transcription of ecDNA-amplified genes. We anticipate that this approach will illuminate mechanisms of ecDNA-mediated drug resistance, by revealing the sensitivity and population dynamics of ecDNA-containing cells under treatment pressure. Citation Format: Owen Chapman, Sunita Sridhar, Jon D. Larson, Robert J. Wechsler-Reya, Jill Mesirov, Lukas Chavez. Detecting and characterizing extrachromosomal DNA in medulloblastoma at single-cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6214.