bluuebunny/crossref_metadata_2025 huggingface dataset:
MEDB-10. Comparing pediatric medulloblastoma with and without spinal metastasis
Authors: Tang Phua Hwee , Nisa Sameema
Published: June 2022

AIM: To compare pediatric medulloblastoma with and without spinal metastasis

METHODOLOGY: Pediatric medulloblastoma cases from 1999 to 2021 were retrospectively reviewed in this Institutional Review Board approved study. Imaging reports, presence of spinal drop metastases at diagnosis, degree of tumor excision, treatment given and survival status were captured.

RESULTS: Brain and spine imaging at diagnosis was available in 54 medulloblastoma patients with no drop metastasis and in 7 with drop metastasis. Largest tumor dimension at presentation is 4.54 ± 0.94 cm with those with drop metastasis, similar to the 4.43 ± 0.94 cm in those without drop metastasis (p = 0.79). For the 54 medulloblastomas with no drop metastasis, 44 (81%) were completely excised, 9 (17%) partially excised and there was no follow up for 1. For the 7 medulloblastomas with drop metastasis, 3 (43%) of the primary tumours were completely excised, 3 (43%) partially excised and there was no follow up for 1. Post operative chemo/radiotherapy was given to 48 of the 54 with no drop metastasis, not given for 1 with no information available for 5. Chemo/radiotherapy was given to 6 of the 7 with drop metastasis with no information available for 1. At 1 year follow up of the 54 with no spinal drop metastasis at diagnosis , 42 remain tumour free, 3 have tumour, 4 are deceased and 5 are lost to follow up. At 1 year follow up of the 7 with drop metastasis, 2 are free of tumour, 2 have tumour and 3 are lost to follow up. Higher percentage of medulloblastomas without drop metastasis are completely excised (p<0.01). No significant difference between postoperative chemotherapy/radiation rates between groups

CONCLUSION: Most medulloblastomas do not have spinal drop metastasis at diagnosis and complete excision is more frequently in those without drop metastasis.
MEDB-12. Severe developmental abnormalities and proliferative cerebellar lesions induced by combined activity of Wnt signalling and loss of SMARCA4
Authors: Göbel Carolin , Holdhof Dörthe , Schoof Melanie , Schüller Ulrich
Published: June 2022

Almost all medulloblastomas (MB) of the WNT subgroup are characterized by hotspot mutations in CTNNB1, and mouse models have convincingly demonstrated the tumor-initiating role of these mutations as well as the tumor origin in the dorsal brain stem. Around 20 % of WNT MB additionally carry SMARCA4mutations, but the functional role of these alterations is mostly unknown. We therefore amended previously described Blbp-cre::Ctnnb1(ex3)Fl/+mice by the introduction of a floxed Smarca4 allele. In contrast to existing literature, Blbp-cre::Ctnnb1(ex3)Fl/+ mice had a maximum life span of only 17 days, even after breeding into two different genetic backgrounds (C57BL/6J and 129S2/Sv). The mice displayed a severe developmental phenotype including a thinned cerebral cortex, hydrocephalus, missing cerebellar foliation and layering as well as non-proliferative cell accumulations in brain stem and cerebellum. An additional homozygous loss of SMARCA4 even resulted in prenatal death for most mice and caused big proliferative lesions in the cerebellum at embryonal day 14.5. These lesions appear to originate from SOX2-positive progenitor cells in the cerebellar ventricular zone. In a next experiment, cells isolated from this region will be characterized in vitro and will be transplanted orthotopically to evaluate their neoplastic potential in vivo. Altogether, we hope to elucidate how a loss of SMARCA4 and mutations of Ctnnb1 cooperate during hindbrain development and tumor formation within this region.
MEDB-15. Dynamic chromatin alteration induces oncogenic hijacking by essential transcriptional factors during SHH medulloblastoma tumorigenesis
Authors: Shiraishi Ryo , Cancila Gabriele , Kumegawa Kohei , da Silva Patricia , Chapman Owen , Wanchen Wang , Jami Maki , Hoshino Mikio , Pfister Stefan , Chavez Lukas , Maruyama Reo , Ayrault Olivier , Kawauchi Daisuke
Published: June 2022

Medulloblastoma is a malignant brain tumor that occurs in the cerebellum, most frequently in children. Medulloblastoma is molecularly classified into four major groups, and therapies are now being developed according to the nature of these groups and subgroups. However, there are currently no effective molecularly targeted drugs for most of these groups. In recent years, we have been analyzing the genomes of medulloblastomas to identify genetic mutations involved in tumorigenesis. Among them, mutations in chromatin modifiers are frequently detected in medulloblastoma, suggesting the importance of alterations in the epigenome in tumor development. However, it remains unclear how epigenomic changes are involved in tumorigenesis. Here, we have used the SHH-group medulloblastoma (MB-SHH) mouse model to understand the epigenomic changes during tumor development and identify new therapeutic targets for medulloblastoma. To this end, we utilized an Atoh1-EGFP:Ptch1+/- mice that specifically label cerebellar granule cell progenitors (GNPs) that are known to be the cellular origin of MB-SHH, preneoplastic (PNCs) and tumor cells with EGFP during MB-SHH formation. Following FACS-based collection of EGFP-positive cells, comparative ATAC-seq analysis revealed that the open chromatin regions dynamically changed during transformation of GNPs into PNCs with enrichment of nuclear factor I (NFI) binding motifs. Cut & Tag analysis on these cells showed that NFI proteins bound chromatin regions that become more accessible during tumorigenesis, suggesting that NFI could play an important role in tumorigenesis after the epigenomic changes. Indeed, CRISPR-engineered in vivo somatic deletion of Nfia and/or Nfib prevented hyperplasia formation, confirming their essential role in tumor initiation. Knockdown of either NFIA or NFIB in patient-derived xenograft models also attenuated tumor growth. Thus, our study has uncovered a novel oncogenic mechanism that contributes to the development of MB-SHH tumors via alterations of accessible chromatin and aberrant DNA binding of NFI transcription factors.
MEDB-17. Re-irradiation for recurrent medulloblastoma in a matched cohort: Advantageous especially in patients without resection
Authors: Adolph Jonas E , Tippelt Stephan , Tschirner Sebastian , Gaab Christine , Mikasch Ruth , Mynarek Martin , Rutkowski Stefan , Warmuth-Metz Monika , Bison Brigitte , Pfister Stefan M , Witt Olaf , Pietsch Torsten , Kortmann Rolf-Dieter , Dietzsch Stefan , Timmermann Beate , Fleischhack Gudrun
Published: June 2022

INTRODUCTION: Radiotherapy with craniospinal irradiation (CSI) is an important part of initial treatment for medulloblastoma in most children. Radiotherapy after recurrence is currently not widely used. This analysis aims to evaluate whether re-irradiation (RT2) may show survival benefits. METHODS: Data for patients with recurrent medulloblastomas from the German HIT-REZ studies was gathered. Patients with RT2 at 1st recurrence were matched by propensity score to an equal number of patients without radiotherapy. Matching variables were sex, initial therapy, time to recurrence, metastatic stage and therapy at 1st recurrence and radiotherapy at subsequent recurrences. The matched cohort was analysed regarding PFS and OS after 1st recurrence. RESULTS: From a cohort of 240 pre-irradiated patients, 106 patients were matched. Patients with RT2 showed improved median PFS [21.0 months (95%-CI: 17.5 – 27.6)] and OS [37.5 months (CI: 30.0 – 59.4)] compared to control patients [(PFS: 12.0 months (CI: 8.1 – 17.7) / OS: 20.1 months (CI: 14.5 – 44.8)]. When stratifying by resection at recurrence (36.8% resected), a survival advantage for RT2 was found in patients without resection in PFS [19.6 (CI: 14.9 – 31.5) vs. 8.0 months (CI: 5.4 – 14.4)] and OS [41.9 (CI: 30.0 – 59.4) vs. 13.3 months (CI: 8.1 – 36.7)]. However, no advantage was found after resection [PFS: 22.5 (CI: 17.5 – 50.4) vs. 19.1 months (CI: 14.1 – 34.3) / OS: 32.3 (CI: 27.6 – NA) vs. 48 months (CI: 23.4 – NA)]. CSI was used in 6 patients without differences in survival to focal RT2. Median PFS after first irradiation was 32.5 months, after RT2 20.9 months. No patients with RT2 were alive past 10 years after 1st recurrence.CONCLUSION: Patients with recurrent medulloblastoma show benefits from RT2 in median PFS and OS. However, no advantage for RT2 was found when resection was also applied at recurrence. Cure after treatment with RT2 was not found in our cohort.
MEDB-20. The outcome of medulloblastoma patients in the 2010-2018 period in Children’s Hospital Zagreb
Authors: Jadrijevic-Cvrlje Filip , Rajacic Nada , Jednacak Hrvoje , Grmoja Tonci , Batos Ana Tripalo , Gjurasin Miroslav , Giljevic Jasminka Stepan
Published: June 2022

This study aims to present the key characteristics of the medulloblastoma patients treated in Children’s Hospital Zagreb and the University Hospital Center Zagreb in Croatia between 2010-2018 period. Croatia has around 145 newly diagnosed pediatric oncology patients annually, including approximately 30 neurooncology patients. We have conducted the retrospective analysis of the hospital records and have collected data on 32 medulloblastoma patients (9 females, 23 males). At the time of diagnosis, the median age was 5,62 (range 0.85-15.86). Before the treatment commencement, we determined conventional risk factors and stratified our patients into standard and high-risk groups (17 standard risk patients, 15 high risk). Qualification for high-risk included metastatic disease, postoperative local residual disease greater than 1.5 cm2, confirmed myc/nmyc amplification in the tumor tissue, and the large cell/anaplastic tumor subtype (p53 positive). The methods of molecular diagnostics were not available at the time. The patients that received solely postoperative chemotherapy were younger than three years. Children younger than five suffering from desmoplastic tumor subtype also received intraventricular methotrexate (Ommaya). High-dosage chemotherapy with autologous stem cell transplantation failed to treat metastatic infant medulloblastoma (2 patients with a lethal outcome). The rest of the patients received craniospinal irradiation, followed by adjuvant chemotherapy. According to the Kaplan-Meier survival analysis, the 5-year overall survival is 65,6 % (40% in the high-risk group and 88% in the standard-risk group). In addition, 5-year event-free survival is 59,4 % (33% in the high-risk group and 82,4% in the standard-risk group). None of the patients developed a secondary malignant disease during the follow-up. Conventional characteristics that determine standard-risk group affiliation are reliable, leading to a satisfactory treatment outcome. The results of the high-risk group treatment are poor necessitating modification treatment approach within clinical trials.
MEDB-21. SOX2+ cells: the perpetrators of medulloblastoma relapse
Authors: Swiderska-Syn Marzena , Mir-Pedrol Julia , Rodriguez-Blanco Jezabel
Published: June 2022

Pediatric brain tumors are the number one cause of cancer-related death in children, with medulloblastoma being the most common type. While survival in patients with medulloblastoma has dramatically improved since chemotherapy was added to standard of care protocols, still 30% of tumors will recur. As recurrent disease in medulloblastoma patients in considered uniformly lethal, it is key to identify the cells allowing tumor relapse, and their targetable regulators. By analyzing single cell transcriptomic data, we uncovered a population of SOX2 labeled astrocyte like cells resistant to SMO inhibitors in clinical trials. Using SOX2-enriched medulloblastoma cultures, we observed that SOX2+ cells rely on non-canonical GLI signaling to propagate medulloblastoma. Therefore, in vivo inhibition of SHH signaling using functionally different GLI inhibitors depleted the SOX2+ cell pool, what led to less aggressive tumors that lacked the ability to further engraft. Stressing the translational relevance of our findings, a clinically relevant GLI inhibitor not only exhausted SOX2+ cells driving tumor relapse, but increased overall survival in mice harboring medulloblastoma. Our results emphasize the importance of using targeted therapies that deplete SOX2+ cells to prevent medulloblastoma recurrence.
MEDB-24. Tumour secreted extracellular matrix predicts survival and influences migration and cell death in SHH medulloblastoma 3D models
Authors: Johnson James , Linke Franziska , Merry Cathy , Coyle Beth
Published: June 2022

Medulloblastoma is the most common malignant paediatric brain tumour. Four molecular sub-groups exist (WNT, Sonic hedgehog (SHH), Group 3 and Group 4), each associated with different patterns of metastasis and chemoresistance. We have shown that within these sub-groups further clinically relevant sub-types exist, characterised by differential expression of extracellular matrix (ECM) proteins. For example, good overall survival in two-thirds of SHH sub-group patients is associated with the expression of specific ECM proteins. Our aim here is to further characterise these ECM components in SHH medulloblastoma and to determine how they confer better overall survival in this sub-group. Using a combination of 3D OrbiSIMs and immunohistochemical staining we have identified, that when grown in 3D hyaluronic acid hydrogels or as spheroids, SHH medulloblastoma cell lines form an ECM shell-like structure composed of laminin, collagen and lumican. In addition, scRNAseq of SHH hydrogel nodules revealed sub-group specific clusters of cells with high levels of ECM interaction and adhesion. We therefore hypothesise that ECM interaction restricts SHH tumour invasion and metastasis through this shell-like structure. To understand how this ECM shell-like structure potentiates better survival in SHH medulloblastoma we have created a CRISPRcas9 laminin knockout SHH medulloblastoma cell line. 3D culture of the laminin knockout cell line demonstrated that laminin is essential for the formation of 3D cell structures as well as migration in SHH medulloblastoma. Furthermore, we have identified that apoptosis is increased in the laminin knockout SHH cell line, suggesting that apoptosis-targeted therapeutics may represent a beneficial treatment option for SHH patients whose tumours exhibit this ECM shell. In summary, tumour-secreted ECM plays a major role in SHH medulloblastoma progression. Expression of laminin, collagen or lumican can be used to classify SHH patients into low and high-risk groups with different therapeutic outcomes and treatment options.
MEDB-32. Reducing treatment-related toxicity for children with WNT-activated medulloblastoma
Authors: Taylor Jessica , Toker Joseph , Masih Katherine , Nathan Erica , Terranova Sabrina , Gilbertson Richard
Published: June 2022

WNT-medulloblastoma has an excellent prognosis, with an overall survival rate of 90% among children receiving standard-of-care (SOC) surgical resection, radiotherapy, and chemotherapy. Unfortunately, while curative, this treatment is associated with major, long-term, debilitating motor, developmental, and neuroendocrine side effects. Therefore, it is crucial we develop effective, less toxic therapies for these children. Similarities have been demonstrated between cancer cell lysosomes and those of patients with Niemann-Pick, a lysosomal storage disease characterised by lysosomal fragility and sphingomyelin accumulation. A class of drugs known as Functional Inhibitors of Acid Sphingomyelinase (FIASMAs), increase lysosomal sphingomyelin and destabilise the cancer cell’s more fragile lysosomal membrane which leads to the induction of cell-death pathways via lysosomal membrane permeabilisation. Loratadine, an antihistamine with high FIASMA activity, consistently induced lysosomal membrane permeabilisation, leading to increased cell-death, in our panel of mouse and human WNT-medulloblastoma lines. Loratadine exhibited no detrimental effect on normal mouse embryonic stem cells from the lower rhombic lip – the putative cell of origin in WNT-medulloblastoma. Luciferase-expressing mouse WNT-medulloblastoma cells were orthotopically implanted into CD1-nude mice and monitored for tumour development via bioluminescent imaging. Upon tumour engraftment, mice were subjected to reduced SOC (radiotherapy and adjuvant vincristine) plus a clinically relevant dose of loratadine. Response and survival were compared to mice treated with full SOC (radiotherapy, vincristine, cisplatin, and etoposide). Mice treated with 2mg/kg/day of loratadine following reduced SOC demonstrated increased survival when compared to those treated with full SOC (p=0.02) along with a significant reduction in weight loss during treatment (p=<0.0001). This work suggests that loratadine, or other FIASMA compounds, may be good alternative adjuvant therapies for WNT-medulloblastoma. Using less toxic adjuvants could improve long-term outcomes through reducing therapeutic related toxicities for children with this devastating disease.
MEDB-36. Clinical and molecular heterogeneity withinMYC andMYCN amplified medulloblastoma
Authors: Schwalbe Edward , Lindsey Janet , Hill Rebecca , Crosier Stephen , Ryan Sarra , Williamson Daniel , Kool Marcel , Milde Till , Pfister Stefan , Bailey Simon , Clifford Steven
Published: June 2022

MYC and MYCN are the most commonly amplified oncogenes in medulloblastoma. Their overall association with a poor prognosis has supported their adoption as high-risk disease biomarkers in trials. However, emerging evidence suggests that certain patients with MYN/MYCN focally-amplified tumours can achieve long-term survival and therefore may suffer unnecessary late-effects associated with intensified therapies. To investigate this heterogeneity, we characterised the molecular and clinico-pathological features of curated cohorts of MYC (n=64) and MYCN (n=95) amplified tumours, drawn from >1000 diagnostic cases, and assessed their associations with disease outcome. Within the MYCN-amplified cohort, survival was related to molecular group; patients with MYCNGrp3 or MYCNGrp4 tumours with no other clinico-pathological risk factors (subtotal resection (STR), metastatic disease, LCA pathology) were intermediate-risk (n=25;70% 5-year PFS). In contrast, a very-high-risk group was defined by positivity for MYCNSHH, STR and/or LCA (n=64;32% 5-year PFS). 22/35 assessable MYCNSHH harboured TP53 mutations; 9/12 with data were germline. MYCGrp3 represented the majority (46/58; 79%) of molecularly-grouped MYC-amplified tumours. Importantly, while radiotherapy receipt conferred a modest survival advantage, for MYC-amplified tumours with additional clinico-molecular risk factors (LCA, metastasis, STR, Grp3), survival was dismal, irrespective of radiotherapy receipt. A very-high-risk group of MYC-amplified tumours was identified (n=51;10% 5-year PFS), defined by positivity for ≥1 additional risk factors (STR, LCA and/or metastasis). Alternatively, membership of subgroups II/V defined a smaller, very-high-risk patient group (n=28;7% 5-year PFS). Long-term survival was seen in the majority of remaining MYC-amplified tumours negative for these specified features (61% 5-year PFS; high-risk). MYC and MYCN-amplified medulloblastomas are biologically heterogeneous with diverse clinical outcomes. Molecular subgroup assignment and established clinical features are critical for their improved stratification. Patient subgroups identified may be eligible for therapy de-escalation; in contrast, the very-high-risk patient groups are incurable using current therapies and urgently require novel experimental treatment strategies upfront.
MEDB-39. Oncogenic mechanisms underlying GLI2-amplified medulloblastoma
Authors: Murad Najiba , Zhang Jiao , Liu Xiao , Tao Ran , Taylor Michael , Pei Yanxin
Published: June 2022

Medulloblastoma (MB) is the most common malignant brain tumor in children. There are several subtypes of MB, and among them, the subtype of GLI2-amplified SHH-MB associated with P53 mutations has the worst prognosis and a poor survival rate; the 5-year survival rate is <30%. Moreover, the GLI2-amplified MBs are non-responsive to the only targeted treatment option available for SHH-MB, the SMO inhibitors. This leaves an unmet critical treatment gap, and there is an urgent need to identify novel targets to develop effective therapeutics. However, a deeper understanding of the cellular and molecular mechanisms driving GLI2-amplified MB tumorigenesis is currently lacking. With a focused goal to resolve this particular type of MB tumorigenesis, we recently generated an engineered mouse model of GLI2-driven MB. Using this model, we demonstrated that GLI2 is the critical driver of tumorigenesis and identified granule cell progenitors (GCPs) as the cells of origin. Interestingly, we have also found that GLI2 drives only Math1+ embryonic GCPs but not neonatal GCPs to form SHH-MB. Correspondingly, our scRNA-seq analysis revealed that the MAPK pathway is specifically enriched in embryonic but not neonatal Math1+ GCPs. Moreover, the MAPK pathway is activated in mouse and human GLI2-driven MB tumors, and a MEK/ERK inhibitor significantly delayed the growth of GLI2-driven MB in vivo. Based on these exciting results, we hypothesize that GLI2-driven MB originates from a specific cell population of Math1+ GCPs and in a particular spatiotemporal window during cerebellar development, and targeting MAPK/MEK/ERK pathway may represent a novel effective approach to treating GLI2-amplified MB.
MEDB-40. RUNNING FOR INCLUSION IN SIOPE PNET5 MB
Authors: Massimino Maura , Boschetti Luna , Minasi Simone , Erbetta Alessandra , Chiapparini Luisa , Mastronuzzi Angela , Miele Evelina , Barra Salvina , Scarzello Giovanni , Cavatorta Claudia , Antonelli Manila , Gandola Lorenza , Buttarelli Francesca Romana
Published: June 2022

Enrolling medulloblastoma(MB) patients in the PNET5 protocol is a daily problem in Italy; since June 2015, 59 cases have been enrolled in 13 centres. So far, 44 of the 103 patients claiming for eligibility did not enter the protocol: 13 metastases, 5 for residual, 20 having exclusion criteria, 4 insufficient frozen material, 2 failure to comply with the correct procedures. No case was lost due to delayed centralization, which is respected even with committing weekends; review of the radiation plan was performed on Saturday for 2 cases, and radiotherapy began on the same day. We made some procedural changes to meet expected deadlines; each local centre notifies the national coordinator of a possible case's existence at MRI diagnosis, of the expected surgery date as well as its realization. MRI imaging is reviewed within 2 days after centralization. Paediatricians notify the national coordinator and pathology/biology reference centre of the MB diagnosis; the shipment of frozen tissue, blood and FFPE is booked. A slot is reserved to priority perform the central pathology review, as well as central molecular diagnosis of genetically defined subgroup (WHO classification) upon receipt of the frozen material. Upon receipt of the FFPE and frozen material, the national reference centre undertakes a double-check with the national coordinator and the local treatment centre to validate the eligibility. Within the 7th day from the receipt of the material: IHC, MYC/MYCN, Monosomy 6, beta-catenin mutation and methylation array are performed. Priority execution of somatic (blood control) sequencing of the PTCH, SUFU, and TP53 genes is also triggered for SHH-activated MB, with the deadline on the 15th day. So far we have had 99% agreement between molecular subgrouping and methylation array. CONCLUSIONS: PNET5 requirements are multiple and changing over time; difficulties may and must be overcome by mutual fast collaboration.
MEDB-41. Identifying a subgroup of patients with early childhood sonic hedgehog-activated medulloblastoma with unfavorable prognosis after treatment with radiation-sparing regimens including intraventricular methotrexate
Authors: Tonn Svenja , Obrecht Denise , Sill Martin , Spohn Michael , Milde Till , Pietsch Torsten , Bison Brigitte , Juhnke Björn-Ole , Struve Nina , Friedrich Carsten , von Bueren André O , Gerber Nicolas U , Benesch Martin , Jäger Natalie , Kool Marcel , Korshunov Andrey , Schüller Ulrich , Pfister Stefan M , Rutkowski Stefan , Mynarek Martin
Published: June 2022

PURPOSE/METHODS: Clinical and molecular risk factors in 142 patients <5 years with desmoplastic medulloblastoma (DMB) or medulloblastoma with extensive nodularity (MBEN) were investigated. Patients were diagnosed between 1992 and 2020 and treated with radiation-sparing approaches, 131 with intraventricular methotrexate. 14 patients with metastatic disease received high-dose chemotherapy. DNA methylation profiles of 77 sonic hedgehog (SHH)-activated medulloblastoma were reclassified according to the Heidelberg Brain Tumor Classifier Version 12.3. RESULTS: While metastatic disease or incomplete resection did not impact progression-free survival (PFS) and overall survival (OS), patients with MBEN had superior outcomes to DMB (5-year PFS 93% vs 71%, p=0.004; 5-year OS 100% vs 90%, p=0.026). Older patients had less favorable PFS (5-year PFS [>3 years] 47% vs 85% [<1 year] vs 84% [1-3 years], p<0.001). No TP53 mutations were detected (n=47). DNA methylation classification identified three subgroups: SHH-1v12.3 (n=39), SHH-2v12.3 (n=19), and SHH-3v12.3 (n=19), with distinct cytogenetic profiles (chromosome 2 gains in SHH-1v12.3, very few alterations in SHH-2v12.3, and chromosome 9q losses in SHH-3v12.3), age profiles (median age [years] SHH-1v12.3: 1.7, SHH-2v12.3: 0.9, SHH-3v12.3: 3.0, p<0.001), and histological distribution (SHH-2v12.3: 74% MBEN, SHH-1v12.3/SHH-3v12.3: 77%/79% DMB, p<0.001). PFS was more unfavorable in patients with SHH-3v12.3-medulloblastoma (5-year PFS 53% vs 86% [SHH-1v12.3] vs 95% [SHH-2v12.3], p=0.002), which remained the only risk factor on multivariable Cox regression for PFS. OS was comparable (5-year OS 94% [SHH-3v12.3] vs 97% [SHH-1v12.3] vs 100% [SHH-2v12.3], p=0.6). 8/9 patients with SHH-3v12.3-medulloblastoma received radiotherapy at relapse (6 craniospinal, 2 local [1 Gorlin syndrome, 1 BRCA2 germline mutation], 1 no radiotherapy [Gorlin syndrome]). CONCLUSION: We identify patients with an increased risk of relapse when treated with radiation-sparing approaches among children with early childhood SHH-medulloblastoma. If these tumors differ from SHH-3-medulloblastoma typically described in older children remains to be verified. Treatment recommendations need to consider cancer predisposition syndromes.
MEDB-47. CD4+ T cells restrict medulloblastoma growth and dissemination
Authors: Eisemann Tanja , Wechsler-Reya Robert
Published: June 2022

The immune system serves as a powerful defense not only against pathogens and parasites but also against neoplastic cells. Emerging immunotherapies that boost the activity of tumor-reactive immune cells or counteract immune suppressive mechanisms have shown promising effects in certain cancer types. However, the success of immunotherapy for brain tumors has been limited, highlighting the need for a better understanding of the immune microenvironment. Our preliminary studies have shown that T cells critically affect tumor growth in mouse models of the pediatric brain tumor medulloblastoma. In particular, depletion of CD4+ T cells results in more aggressive growth of medulloblastoma cells and allows these cells to metastasize to the spinal cord. To test whether CD4+ T cells can recognize and attack tumor cells directly, we generated MHC class II knockout tumors. Surprisingly, depletion of CD4+ cells still enhanced tumor growth and metastasis. These results suggest that CD4+ T cells regulate medulloblastoma growth independently of MHC II on tumor cells. We hypothesized that CD4+ T cell may not directly kill tumor cells but recruit and activate another effector immune cell type that eliminates tumor cells. As CD4+ T cells have a well-studied helper function for CD8+ T cells, we examined whether their anti-tumoral function relies on the activation of cytotoxic CD8+ T cells. The depletion of CD4+ T cells still resulted in advanced growth of MHC class I-deficient, and thus CD8+ T cell resistant, tumor cells indicating that CD4+ T cells counteract tumor growth in a CD8+ T cell-independent manner. Ongoing studies are aimed at elucidating the mechanisms by which CD4+ T cells regulate medulloblastoma growth, including the antigen-presenting cells that activate them and the effector cells responsible for killing tumor cells. These studies will advance our understanding of the immune microenvironment in medulloblastoma and allow us to design more effective therapies.
MEDB-52. Organoids as preclinical models to improve and personalize disease outcome for sonic hedgehog medulloblastoma
Authors: Odé Zelda , Maas Joris , Roosen Mieke , Stathi Phylicia , Federico Aniello , Mack Norman , Schwalm Benjamin , Bunt Jens , Kool Marcel
Published: June 2022

Four main medulloblastoma (MB) molecular subgroups are known, including the sonic hedgehog (SHH) subgroup, which represents ~25% of MB cases. The 5-year overall survival of SHH-MB is ~80%. However, survival between patients is highly diverse and dependent on the driver mutation(s) of the tumor. Patients with TP53 mutated tumors (often accompanied with MYCN and/or GLI2 amplifications) don’t respond well to current therapies and have a 10-year overall survival below 20%. Therefore, there is a need for new and more tailored therapies for these patients. In this study we aim to screen patient-derived organoid models of TP53-mutated SHH MB with a library of ~200 different compounds. We have optimized the cultures of two PDX-derived and one patient-derived organoid line in vitro. The lines will be screened in a high-throughput manner and the best hits and combinations will be validated in corresponding in vivo PDX models. To further assess the role of specific mutations in therapy outcome of TP53-mutated SHH MB, cerebellar organoids generated from human iPSCs were genetically modified with overexpression of dominant-negative P53 (DNP53) alone or in combination with MYCN and/or GLI2. Introduction of DNP53 and MYCN overexpression in cerebellar organoids at day 28/35 leads to the outgrowth of a Ki67-positive proliferating mass after three weeks, indicating tumor growth. Further analyses are ongoing to see how they match SHH-MB patient tumors. These genetically engineered organoid models may elucidate the role of specific mutations in therapy response and/or resistance. In addition, as tumors in these genetically engineered cerebellar organoids arise in a microenvironment of normal cerebellar cell types, initial safety of drugs on cerebellar cells can be assessed. In conclusion, different organoid models of TP53-mutated SHH MB will enable us to find more effective treatments and to better understand how to treat patients with different mutation combinations.
MEDB-53. Incidence of Hearing impairment in childhood Medulloblastoma Survivors treated in King Fahad Medical City KFMC Saudi Arabia
Authors: Mahmodi Mohammed Al , Mobark Nahla Ali , Almowanes Demah A , AlGhunaim Haya N , Kattan Nad E , Aljabarat Wael abdel Rahman , Alotabi Fahad , Rayis Mohammed , AlNaqib Zaid G , Balbaid Ali Abdullah O , Alharbi Musa
Published: June 2022

Medulloblastoma MB is the most common childhood CNS tumor treated with intensive multimodalities therapy Surgery, Radiation and Cisplatin chemotherapy which causing significant Hearing impairment (HI) with profound impact on child’s quality of life. METHOD: Retrospective study evaluating risk factors & (HI) incidence in childhood MB survivors treated in KFMC between 2010 and 2020. (HI) was graded using the National Cancer Institute (NCI) Grades (1-4) ALL patients received 6 weeks of Risk adapted CSI radiotherapy concurrent with daily oral Etoposide. patients were treated with 2 different Maintenance chemotherapy German HIT-MED MB protocol 8 Maintenance cycles (Cisplatin 70 mg/m² x 1d /lomustine/VCR) Cumulative dose of Cisplatin 560 mg/m2 KFMC MB SAPHOS protocol 6 Maintenance cycle alternating A&B. cycle A: Cisplatin 90 mg/m² x 1d & 3 weeks of daily oral Etoposide. cycle B: Cyclophosphamide x 2 d & Vincristine Cumulative dose of Cisplatin 270 mg/m2. RESULTS: At Median follow-up of 5 years total of 78 MB survivors, male predominance 66.7% Median age at diagnosis 82 months post end of therapy 28.2% maintain normal hearing while 71.8% had hearing decline compared with baseline audiology Median time of onset of hearing decline was 7 months after start of radiation therapy Significant (HI) (NCI grade 3/4) in 26.9% of patients, and 23.1% required hearing aid use NO statistically difference in incidence of significant (HI) in patients treated with HIT-MED protocol (26.7%) & patients treated with KFMC MB-SAPHOS protocol (27.1%) OR those treated as Average-Risk CSI radiotherapy 22.9% Vs High-Risk CSI radiotherapy (30.2%), patients age at diagnosis (< or > 5 years) does not affects HI incidence. CONCLUSIONS: significant hearing impairment incidence was not affected by Radiation doses OR cumulative Cisplatin dose. Our study will form base line for future studies to modify therapy related Toxicity and improve outcome of childhood MB in Saudi Arabia.
MEDB-59. A draft atlas of medulloblastoma cellular evolution under therapy
Authors: Wang Lin , Jung Jangham , Babikir Husam , Shamardani Karin , Kasahara Noriyuki , Müller Sabine , Diaz Aaron
Published: June 2022

How standard care shapes the cellular composition of recurrent medulloblastoma (MB), if therapy selects for specific tumor or immune cell types, is unknown. We report the pilot phase of our ongoing effort to profile human longitudinal MB specimens via single-cell transcriptomics and epigenetics. We profiled 11 diagnostic and eight recurrent specimens from 19 subjects via single-nucleus RNA sequencing (snRNA-seq), and four subjects via single-nucleus assay for transposase-accessible chromatin. Specimens from select subjects were also profiled to assess genome-wide enhancer activity via single-nucleus cleavage-under-targets and tagmentation. We found an upregulation of the DNA-damage response, RNA translation, WNT and NOTCH signaling in recurrent specimens. The percentages of stem-like cells increased by over two-fold at recurrence. We found that microglia and oligodendrocyte-lineage cells were the most abundant non-malignant tumor-associated cell types, representing 2%-10% of cells profiled. Microglia abundances were relatively stable across molecular subtypes, and when comparing primary to recurrent tumors. There was a moderate, but statistically significant, increase in oligodendrocyte abundance in SSH and WNT tumors, compared to Group 3/4 tumors. We compared gene expression in tumor cells with public snRNA-seq from developing human cerebella (PCW 9-21). Combined Group-3/4 cell analysis supports a common lineage hierarchy, with an enrichment for unipolar brush-cell and Purkinje-cell phenotypes found in Group-4 tumors. All Group-3/4 cases contained cycling cells expressing markers of PAX2+ interneuron progenitors, most cycling cells had this phenotype. All specimens contained populations of non-cycling granule-cell progenitor-like cells. We performed single-cell co-expression receptor/ligand analysis to infer paracrine signaling between tumor and non-malignant cell types. This identified both tumor cells and microglia as sources of growth factors, pro-inflammatory cytokines, and pro-apoptotic ligands. Non-malignant oligodendrocyte-lineage cells uniquely expressed IL6-family cytokines, pleiotrophin, and class-III semaphorins. These studies shed light on the cellular heterogeneity of MB and the effect of standard therapy in shaping composition at recurrence.
MEDB-61. Genetic alterations ofTP53 andOTX2 indicate increased risk of relapse in WNT medulloblastomas
Authors: Goschzik Tobias , Mynarek Martin , Dörner Evelyn , Aretz Stefan , Rutkowski Stefan , Pietsch Torsten
Published: June 2022

PURPOSE: This genetic analysis of WNT-activated medulloblastomas (WNT-MBs) aimed to re-evaluate the prognostic impact of TP53 mutations and to identify specific chromosomal aberrations as possible prognostic markers in a retrospective cohort of patients treated according to the protocols of the HIT medulloblastoma studies. PATIENTS AND METHODS: In a cohort of 191 patients with WNT-MBs, mutations in CTNNB1, APC, and TP53 were analyzed by Sanger and/or NGS panel sequencing. Chromosomal copy number aberrations (CNAs) were assessed by high-resolution, genome-wide molecular inversion probe technology (MIP), SNP6 array, and/or 850k methylation bead-array hybridization. Complete clinical data were available from 120 patients. RESULTS: Patients with WNT-MBs had a female predominance (1.4:1) and a median age of 13 years (range 3-69 years). CTNNB1 mutations were present in 92.2% of the samples, APC mutations in 6.8%. One CTNNB1 wildtype tumor gained WNT-activation due to a homozygous deletion of FBXW7. Monosomy 6 was present in 78.6%, but more frequent in children compared to adults. 16.1% of the tumor samples showed TP53 mutations, of those 60% with nuclear positivity for the p53 protein. A loss of heterozygosity at the TP53 locus on chromosome 17p13.1 was found in 40.7% (11/27) of TP53 mutant tumor samples and in 18.5% of the whole cohort (24/130 cases). Patients with tumors harboring TP53 mutations showed significant worse progression-free survival (PFS; p=0.001), but not overall survival (OS) and were enriched for chromosomes 17p (p=0.001), 10, and 13 losses. Gains of the OTX2 locus on chromosome 14q were also associated with poor PFS and OS (p=0.017 resp. p=0.006). Multivariate Cox regression analysis identified both genetic alterations as independent prognostic markers for PFS and OS. CONCLUSION: For ongoing and future de-escalation trials for patients with WNT medulloblastomas, we recommend to exclude patients with tumors carrying TP53 mutations or OTX2 gains.
MEDB-67. Subgroup specific analysis of cellular metabolism in medulloblastoma
Authors: Funke Viktoria , Walter Carolin , Melcher Viktoria , Wei Lanying , Sandmann Sarah , Varghese Julian , Jäger Natalie , Albert Thomas K , Schüller Ulrich , Kerl Kornelius
Published: June 2022

INTRODUCTION: Molecular subgrouping of Medulloblastoma (MB) has expanded our understanding of its biology and the impact on clinical parameters. However, detailed analysis of inter- and intratumoral heterogeneity on a metabolic level is currently lacking. Within this study, we aimed at improving our understanding of metabolic heterogeneity between the MB subgroups, between samples within these subgroups and how these differences affect prognosis. METHODS: We analyzed metabolic characteristics of four MB cohorts covering 1,804 samples in total. In 911 samples (ICGC and MAGIC cohort), we explored metabolic programs on RNA level. In two cohorts (ICGC and G3/G4 samples from the HIT cohort; n=1,035) we examined genetic alterations on DNA level. Furthermore, single-cell RNA-sequencing data of six samples were used to explore intratumoral metabolic heterogeneity. Inter- and intratumoral heterogeneity were correlated to clinical data. RESULTS: Using publicly available gene signatures, we discovered significant differences in metabolic gene expression comparing established MB subgroups. Three metabolically distinct clusters of G3/G4 samples could be defined by unsupervised analyses in two independent cohorts. We were able to confirm our finding of intertumoral metabolic differences on single-cell RNA level. Additionally, our analysis revealed the possibility of sample-specific metabolic features. On DNA level, we identified regulatory genes with known role in MB development to be predominantly associated with lipid metabolic processes. After all, lipid metabolism and metabolism of nucleotides in MB have prognostic value and correlate with the outcome of patients. CONCLUSION: Our data highlight the importance of metabolic properties in MB. We show the distinct metabolic signatures are clinically relevant and, thus, might provide opportunities for novel target-directed therapeutic options in the future.
MEDB-73. Lipid metabolism as a therapeutic vulnerability in BET inhibitor-resistant medulloblastoma
Authors: Lupien Leslie , Boynton Adam , Chacon Madison , Kumbhani Rushil , Gionet Gabrielle , Goodale Amy , Root David , Keshishian Hasmik , Robinson Margaret , Carr Steven , Bandopadhayay Pratiti
Published: June 2022

MYC-driven medulloblastomas are a particularly devastating group of pediatric brain tumors that exhibit resistance and continued progression despite standard of care treatments. Our preclinical work identified BET-bromodomain inhibitors as a potentially promising new class of drugs for children with medulloblastoma and other MYC-driven cancers, providing rationale to evaluate these agents in clinical trials. However, treatment with BET inhibitor (BETi) alone is unlikely to be sufficient to cure, with most tumors evolving to acquire resistance to single-agent targeted therapies. We applied an integrative genomics approach to identify genes and pathways mediating BETi response in medulloblastoma. These studies revealed that MYC-driven medulloblastoma cells with acquired resistance to BETi reinstate transcription of essential genes suppressed by drug and exhibit changes in cell state and new vulnerabilities not present in drug-sensitive cells. We now have a growing body of evidence showing that BET inhibition downregulates the expression of key lipid metabolism genes and metabolism-related signaling pathways, and that medulloblastoma cells with adaptive resistance to drug differentially express and exhibit preferential dependency on specific lipid metabolic genes and transcriptional regulators. Our studies explore the possibility of exploiting these metabolic vulnerabilities to overcome BETi resistance and provide a more efficacious upfront therapy.
MEDB-81. Combined inhibition of CDK11 and EZH2 results in regression of MYC-amplified medulloblastoma
Authors: Wang Dong , Veo Bethany , Pierce Angela , Venkataraman Sujatha , Vibhakar Rajeev
Published: June 2022

We explored an shRNA library screen on 20 cyclin-dependent kinases to establish cyclin-dependent kinase 11 (CDK11) as a critical mediator in MYC-driven medulloblastoma. The effect and molecular mechanism of CDK11 in the proliferation and growth of medulloblastoma were investigated in vitro. Pharmaceutic inhibitors and genetic depletion of CDK11 resulted in cessation of tumor growth in xenograft mouse models. Through combination chemical screening, we identified that 5-FU enhanced the apoptosis which induced by inhibition of CDK11 in medulloblastoma cells. In addition, we found CDK11 is a significant candidate kinase participating in the negative control of Wnt/b-catenin signaling. Down-regulation of CDK11 led to the accumulation of Wnt/β-catenin signaling receptor complexes through activation of transmembrane Frizzled (FZD) receptors which is suppressed by H3K27Me3. RNASeq and cut&run revealed that Cdk11 and mediator associated Cdk8 kinase regulate a common set of genes. Lack of Cdk8 and Cdk11 impaired Ezh2 recruitment and the establishment of histone H3 lysine 27 tri-methylation. We concluded that combined EZH2 and CDK8/CDK11 inhibitors treatment concurrently activated Wnt signaling may be an effective treatment for Group 3 medulloblastoma.
MEDB-83. A novel epigenetic nanotherapeutic strategy to induce medulloblastoma differentiation
Authors: Raju Praveen , Tylawsky Daniel , Vaynshteyn Jake , Gerwin Jeffrey , Heller Daniel , Snuderl Matija
Published: June 2022

The histone-lysine N-methyltransferase EZH2 is the catalytic component of the PRC2 complex and is overexpressed in several medulloblastoma subtypes. However, its role in medulloblastoma tumorigenesis has been shown to be context-dependent using genetic approaches. Furthermore, pharmacological approaches have been limited by the very poor blood-brain barrier (BBB) penetration of current EZH2 inhibitors in use. Using laser capture microdissection and RNA-Seq analysis of human nodular/desmoplastic SHH medulloblastoma FFPE tissue, we provide data for the spatial epigenetic heterogeneity of primitive/proliferative regions compared to nodular/mature regions. Bioinformatic analysis identifies ~120 differentially expressed genes between primitive and mature regions with enrichment for genes regulated by H3K4me3 and H3K27me3 or SUZ12. ChIP-Seq analysis shows striking differences in H3K27me3 enrichment between primitive and mature medulloblastoma cells including at the EZH2 locus. Utilizing a genetically-engineered mouse model of SHH medulloblastoma, we show that conditional EZH2 genetic ablation within medulloblastoma cells results in wide-spread tumor cell differentiation (n=31 mice; *p=2e-07). Conversely, conditional EZH2 (Y641F) activation in this GEM model prevents tumor cell differentiation. Notably, we have found that the CDNK2A (p16) locus is an important EZH2 target that regulates tumor cell differentiation. qRT-PCR analysis of SHH medulloblastoma in wild-type and Ezh2 knockout settings show significant reduction in Gli1 and CCND1 and increase p15 and p16 expression in Ezh2 knockout mice compared to Ezh2 wildtype mice (*p<0.05). Importantly, genetic ablation of p16 conditionally in SHH MB EZH2 double knockout mice rescues the widespread tumor cell differentiation (n=9 mice; *p=3e-06) seen in Ezh2 single knockout SHH medulloblastoma mice. Finally, we developed a novel fucoidan-based nanoparticle strategy to deliver the EZH2 inhibitor (EPZ-6438) across the intact BBB of this GEM model to achieve significant extension of mouse survival (median 70 days compared to 19 days in control mice; *p=0.01, Mantel-Cox) with potential utility for other pediatric brain tumors.
MEDB-90. Iron Imbalance Can Potentiate Cisplatin Response in Pediatric Medulloblastoma by Regulating Ferroptosis
Authors: Kanchan Ranjana , Perumal Naveenkumar , Khan Parvez , Doss David , Venkata Ramakanth Chirravuri , Thapa Ishwor , Vengoji Raghupathy , Kaushal Jyoti , Siddiqui Jawed , Nasser Mohd Wasim , Batra Surinder , Mahapatra Sidhartha
Published: June 2022

Medulloblastoma (MB), the most common malignant pediatric brain tumor, is a leading cause of childhood mortality. Of the four primary subgroups, patients with group 3 tumors have the poorest prognosis. Loss of chromosome 17p is a high-risk feature associated with poor outcomes in group 3 tumors. We recently elucidated the tumor suppressive properties of a novel miR, miR-1253, on the terminal end of 17p. In further exploring its anti-neoplastic effects, we discovered that miR-1253 can disrupt iron homeostasis, causing oxidative stress and inducing lipid peroxidation. These concurrent events are capable of triggering an iron-mediated form of cell death called ferroptosis. Notably, our in silico interrogation of ferroptosis regulator genes (FRGs) in group 3 tumors revealed high expression of genes associated with iron transport and glutathione metabolism. These included mitochondrial iron transporters and GPX4, a critical regulator of ferroptosis. Restoration of miR-1253 expression in group 3 cell lines resulted in specific downregulation of ABCB7, an iron-sulfur cluster exporter, and GPX4. Consequently, cytosolic and mitochondrial labile iron pools rose, glutathione levels declined, and mitochondrial oxidative stress and lipid peroxidation were induced. These events were recapitulated by ABCB7 knockdown and potentiated cell death. Treating miR-1253-expressing cancer cells with cisplatin, a group 3 MB chemotherapeutic agent with ferroptotic properties, further elevated oxidative stress, depleted glutathione levels, and augmented lipid peroxidation, with added inhibitory effects on cell viability and colony formation. Treatment with a ferroptosis inhibitor (ferrostatin-1) lead to recovery from the cytotoxic effects of this combination therapy. Our studies highlight a novel mechanism for group 3 MB pathogenesis via ferroptosis regulation and provide a proof-of-concept for exploiting group 3 MB tumor vulnerability to iron imbalance as a novel treatment strategy.