bluuebunny/crossref_metadata_2025 huggingface dataset:
LGG-01. DEVELOPMENT OF A WEB-BASED CALCULATOR APPLICATION FOR PREDICTING SHORT- AND LONG-TERMSURVIVAL IN PEDIATRIC OPTIC PATHWAY GLIOMA: A POPULATION-BASED DATABASE ANALYSIS
Authors: Brown Nolan , Kuo Cathleen , Pennington Zach , Gendreau Julian , Givens Drew , Daniels David
Published: June 2023

Optic pathway gliomas (OPG) are generally low-grade lesions arising from the astrocytes of the visual pathway, most commonly of the optic nerves and chiasm. Uncertainty remains regarding the optimal management of this patient population. We sought to leverage population-level data from the Surveillance, Epidemiology, and End Results program database to develop a predictive calculator for overall survival (OS) amongst pediatric patients with OPG. A total of 1,052 patients who had been diagnosed with OPG were included. Multivariate cox proportional hazard model demonstrated patients of “other” race (hazard ratio [HR]=4.109, 95% confidence interval [CI] 1.076-15.694, p=0.039) exhibited a decreased overall survival, while patients with a median household income ≥$55,000 (HR=0.172, 95% CI 0.065-0.457, <0.001) and those who underwent subtotal resection (HR=3.549, 95% CI 1.092-13.364, p=0.047) had a higher overall survival rate. An online, interactive survival prediction tool (https://spine.shinyapps.io/optic_glioma/) was created to estimate 5-, 10-, and 15-year overall survival probability as well as median survival months. C-index was 0.722 (95% CI 0.642-0.776) for the training set and 0.745 (95% CI 0.661-0.790) for the validation set. Good concordance between the predicted probability and the actual probability of the dataset was also observed. This calculator may serve as a useful clinical tool during patient counseling. Further investigations aimed at developing calculators of visual acuity preservation and progression-free survival are merited; progress toward this end will benefit from continued multicenter collaborations.
LGG-07. TARGETED THERAPY IN PEDIATRIC LOW-GRADE GLIOMAS; REAL WORLD DATA IN THE CONTEXT OF CONVENTIONAL TREATMENT MODALITIES
Authors: Sumerauer David , Trkova Katerina , Koblizek Miroslav , Kyncl Martin , Krskova Lenka , Benes Vladimir , Zapotocky Michal
Published: June 2023

Progressive inoperable pediatric low-grade gliomas (pLGG) are challenging and chemotherapy provides unsatisfactory long-term disease control. Majority of pLGGs harbor oncogenic MAPK pathway alterations. BRAF and MEK inhibitors were developed and available data suggest possible benefit in pLGG patients. Therefore, we aimed to evaluate efficacy and toxicity of dabrafenib in BRAF-V600E positive pLGG and trametinib in KIAA1549::BRAF fused or NF1-associated pLGGs. Retrospective review of patients with molecularly profiled pLGGs treated with MAPK inhibitors at our institutions was performed. Response to targeted therapies was evaluated using RAPNO criteria and volumetric analysis. Twenty-six patients were eligible for this study because they had either BRAF-V600E pLGG treated with dabrafenib (n=11) or KIAA1549::BRAF fused/NF1-associated pLGG treated with trametinib (n=15) outside clinical trials. Patients received targeted therapy because of radiological progression (n=18), clinical deterioration (n=2) or as an upfront approach (n=6). Overall response rate (ORR) of the whole cohort using RAPNO criteria was 46.1% (12/26). There was a discordance observed between RAPNO bidimensional and manual volumetric analysis in 7 patients (26.9%). Progression free survival (PFS) at 2 years for the whole evaluated cohort was 78.5%(95 CI, 63.2 to 97.4%). ORR of patients with BRAF-V600E pLGG was 64% (median time to response 2.6 months). Out of fifteen patients treated with trametinib, 33% achieved partial response (median time to response 6.6 months). In total, 6 patients experienced progression on therapy. Six patients were treated with vinca alkaloids and trametinib with no additional toxicity. All 15 patients experienced trametinib-related adverse event, grade III was reported in 6 (40%) of them. Altogether 11 patients (73%) required dose reductions or treatment interruptions. MAPK inhibitors represent novel treatment modality in pLGG patients with clinical benefit in proportion of patients. Nevertheless, patients may experience progression on therapy requiring further management.
LGG-08. TARGETED THERAPY IN BRAFV600E-DRIVEN PEDIATRIC GLIOMAS: UNCOVERING MECHANISMS OF RESISTANCE THROUGH A MULTI-OMIC APPROACH
Authors: Capogiri Monica , De Micheli Andrea , Berenjeno-Correa Ernesto , Postlmayr Andreas , Guerreiro Stücklin Ana Sofia
Published: June 2023

BRAFV600E is a key oncogenic mutation, detected in both pediatric low-grade gliomas and high-grade gliomas. Targeting the Ras-BRAF-MAPK pathway with BRAFV600E-specific inhibitors, such as Dabrafenib, is a therapeutic strategy increasingly used in clinical practice. However, therapy failure often emerges in patients, and the mechanisms driving drug resistance remain elusive. We apply a multi-omic approach to understand the evolution of BRAFV600E glioma cell lines and ex-vivo patient-derived cultures under BRAF inhibition. To recapitulate the clinical situation and decipher mechanisms of acquired resistance, we chronically expose BRAFV600E-positive glioma cells to a sublethal dose of Dabrafenib (IC20) and generate drug-resistant cell lines. We then profile the kinome (Pamgene microarray) and the transcriptome (bulk RNAseq) of resistant versus wildtype cells: in addition to reactivation of the Ras-BRAF-MAPK pathway, we observe increased activation and expression of PDGFRB and Src family kinases (SFK) in the Dabrafenib-resistant phenotype. To identify and track the longitudinal evolution of drug-resistant states, we further assess the evolution of glioma cell subpopulations under therapeutic pressure and profile consecutively collected samples (day 0; day 2; day 10; day 30 of treatment; and 10 days post-treatment) using single-cell transcriptomics (10x Chromium system, 10x Genomics). By profiling BRAFV600E glioma cells pre-, on- and post-treatment, we find that therapeutic pressure induces changes in cell subpopulations from early stages, and these changes are selected over time. Consistent with our kinome and bulk RNAseq findings, we confirm the role of PDGFRB in driving Dabrafenib resistance in a subset of cells. Furthermore, when off-treatment, the cells recapitulate the untreated status, indicating that resistance is a temporary, drug-dependent, phenomenon relying on the kinome rewiring. By dissecting the pro-survival role of the PDGFR/SFK axis upon Dabrafenib treatment, we will provide valuable mechanistic and therapeutic insights into targeted therapies for children with BRAFV600E gliomas.
LGG-13. GANGLIOGLIOMA DEEP TRANSCRIPTOMICS REVEALS A CD34+ PRIMITIVE NEUROECTODERM NEURAL PRECURSOR-LIKE POPULATION
Authors: Regal Joshua , Guerra-Garcia Maria , Jain Vaibhav , Chandramohan Vidyalakshmi , Ashley David , Gregory Simon , Thompson Eric , Lopez Giselle , Reitman Zachary
Published: June 2023

Gangliogliomas are glioneuronal brain tumors that typically present in childhood or early adulthood. Though most often low-grade, new insights are needed to refine glioneuronal tumor classification and to identify therapeutic approaches, particularly for unresectable, high-grade, and/or recurrent disease. Gangliogliomas often possess a rare population of immature astrocyte-appearing CD34+ cells. CD34 is expressed in neuroectoderm neural precursor cells during embryogenesis. Neural cell CD34 expression is usually lost prior to birth. We hypothesized that CD34+ ganglioglioma cells represent tumor precursor/stem cells and closely resemble ganglioglioma cells of origin. To test this, we evaluated five gangliogliomas with single nucleus RNA-seq, cellular indexing of transcriptomes and epitopes by sequencing, and/or spatially-resolved RNA-seq. Developmental trajectory analyses uncovered a neoplastic cellular hierarchy, with CD34+PAX6+SOX2+MSI1+PECAM1-VWF-DCN-COL1A2- cells being the most primordial and precursor to neuron-like and macroglia-like neoplastic cell states. Gene regulatory network inference of CD34+ ganglioglioma cells indicated TCF7L2/MEIS1-PAX6 and SOX2-mediated transcriptional programming, similar to that found during neuroectodermal/neural development in utero. Spatially-resolved transcriptomics revealed a neuroectoderm neural precursor-like tumor cell niche relatively devoid of vascular and immune cells. We used these high-resolution results to deconvolute clinically-annotated transcriptomic data, confirming that CD34+ neural precursor-like cell-associated gene programs associate with gangliogliomas compared to other brain tumors. Together, these deep transcriptomic approaches characterized a ganglioglioma cellular hierarchy - identifying CD34+ neuroectoderm neural precursor-like tumor-initiating cells, corresponding regulatory transcriptional programs, cell signaling pathways, and associated immune cell states. These findings may facilitate improved ganglioglioma tumor classification, diagnosis, and therapeutic investigations.
LGG-21. FGFR1 GENE MUTATION AS A POTENTIAL RISK FACTOR FOR SPONTANEOUS INTRACRANIAL HEMORRHAGE IN PEDIATRIC LOW GRADE GLIOMA PATIENTS
Authors: Lebert Brittany , Campion Stephani , Wagner Aaron , Gonzalez-Vega Maxine , Aguilar-Bonilla Ana , Smith Amy
Published: June 2023

The Fibroblast Growth Factor Receptor 1 (FGFR1) gene is the second most altered gene in pediatric LGGs and has been associated with poorer prognoses. A recent study suggested a link between the FGFR1 mutation and spontaneous intracranial hemorrhage in pediatric LGG patients, an event that can be detrimental to cognitive development and, in some cases, even fatal. The current study aimed to further confirm this link by following 49 pediatric LGG patients treated at the Arnold Palmer Hospital for Children from 2007-2022 and investigating the occurrences of spontaneous intracranial hemorrhage. We accessed their treatment course, clinical outcome, radiographic findings, and pathological findings. Genetic analysis was performed as part of their standard of care at the time of tumor resection or biopsy. Of the forty-nine pediatric LGG patients we followed, eight (16.3%) experienced spontaneous intracranial hemorrhage. Five of these eight patients (62.5%) had an FGFR1 mutation. Out of the forty-nine patients, only six of them had an FGFR1 mutation, and five out of the six (83.3%) experienced spontaneous intracranial hemorrhage at some point during treatment. The one patient that did not experience hemorrhage lacked a classic FGFR1 mutation and presented with a variant of unknown significance that has yet to be regarded as pathogenic. These findings are consistent with the aforementioned study and are significant because, to date, little is known about the tumor-specific risk factors for these spontaneous intracranial hemorrhages. Understanding the risk factors could help clinicians better predict these occurrences and potentially prevent their devastating effects.
MDB-03. DFMO AS A TREATMENT STRATEGY IN GROUP 3 MEDULLOBLASTOMA
Authors: Shahab Shubin , Roggeveen Christianna , MacDonald Tobey , Kenney Anna
Published: June 2023

Medulloblastomas, the most common malignant brain tumor in children, are subdivided into 4 major molecular subgroups. Among these, Group 3 medulloblastomas have one of the worst prognoses. We have recently identified the LIN28B pathway as being critically important in Group 3 medulloblastoma growth in vitro and in vivo. There are few targeted inhibitors of the LIN28B pathway and none are currently in clinical practice. Recently DFMO has been identified as an indirect inhibitor of the LIN28B-let-7 axis through its inhibition of polyamine biosynthesis. Another recent study identified ornithine, a precursor to polyamines, as being upregulated in MYC amplified Group 3 medulloblastoma. Importantly, DFMO has been demonstrated to achieve high concentrations in brain tumors in a rat glioma model with minimal systemic toxicity. Based on this we hypothesize that DFMO would be attractive agent for Group 3 medulloblastoma treatment as it would inhibit both polyamine synthesis and LIN28B and potentially achieve high tumor concentrations. Here we demonstrate that treatment with increasing concentration of DFMO leads to significant reduction in Group 3 medulloblastoma viability, increased apoptosis and reduced levels of LIN28B and its downstream targets. We are currently investigating whether this drug can effectively inhibit in vivo tumor growth in orthotopic xenograft models of Group 3 medulloblastoma.
MDB-07. MYC AND TGFΒ PROMOTE GROUP 3 MEDULLOBLASTOMA TUMOR RESISTANCE THROUGH DEREGULATION OF KDM2B TARGETS
Authors: Qadeer Zulekha , Westelman Samantha , Johnson Mackenzie , Grele Shane , Hou Elise , Hendrikse Liam , Wang Linyu , Husain Sarah , Beytagh Mary Clare , Schmidt Christin , Huang Miller , Taylor Michael , Weiss William
Published: June 2023

Medulloblastoma (MB) is one of the most prevalent malignant brain tumors in children, with tremendous cognitive and neuroendocrine disability among survivors. Group 3 (G3) MBs have poor overall survival at <50%, few recurrent mutations, higher frequency of metastasis, and no targeted therapies. Amplification of MYC and activation of TGFβ signaling are frequent in G3MB. Remarkably, some MB tumors have no reported mutations, suggesting roles for epigenetic mechanisms in driving disease. We hypothesize that the TGFβ pathway and MYC contribute to the intrinsic resistance of G3MB through deregulation of key genes and pathways. We previously established humanized models for SHHMB by introducing MYCN or PTCH1 deletions into neuroepithelial stem cells (NESC) derived from normal human induced pluripotent stem cells (hIPSCs). In this study, we transduced NESCs with TGFb effectors activated in G3MB alone and/or in combination with MYC, prioritizing combinations observed in patients. Excitingly, both MYC and TGFβ effectors drove tumor formation in vivo with the combination of TGFβ effectors with MYC leading to more aggressive tumors. We next found that NESCs expressing MYC with either TGFβR1 or TGFβ1 showed resistance to clinical TGFβR1 inhibitors, compared to cells driven by either TGFβR1 or TGFβ1 alone. To decipher mechanisms of resistance, we integrated CUT&RUN to probe for MYC genomic localization and relevant histone PTMs with RNA-seq analysis and discovered a subset of genes upregulated in MYC and TGFb-driven lines that are targets of the histone demethylase KDM2B. Loss of function mutations in KDM2B occur in G3MB patients, indicating that KDM2B deregulated genes are critical in G3MB. We postulate that epigenetic remodeling via MYC and recruitment of other MYC-interacting cofactors to KDM2B targets culminates in transcriptional changes that lead to aggressive disease. Overall, our studies provide important insight on identifying new therapeutic avenues for patients with MYC and TGFβ driven G3MB.
MDB-10. EPIDEMIOLOGICAL PROFILE OF MEDULLOBLASTOMA IN MOROCCO: A 2-YEAR RETROSPECTIVE STUDY
Authors: Erefai Ouassima , Sennani Sara , Soulaymani Abdelmajid , Mokhtari Abdelrhani , Hami Hinde
Published: June 2023

Medulloblastoma is a malignant, invasive embryonal tumor of the central nervous system. This pathology is the leading malignancy in children, accounting for 30% of central nervous system pediatric tumors. This study aimed to present the epidemiological profile of medulloblastoma in Morocco. It was a retrospective study including all children aged 0 to 16 years and diagnosed with medulloblastoma at the pediatric hematology-oncology department of the children’s hospital in Rabat (Morocco) over two years, from January 1, 2021, to December 31, 2022. A total of 12 patients were included in the study, 7 boys and 5 girls (M/F ratio 1.4). The mean age was 9 ± 3 years. The majority (91.7%) of children were over 5 years of age. Only one child was aged 18 months. over half of the patients were from a rural areas. Most cases were living over 30 Km from the center. Among them, 40% were more than 150 km. A low socio-economic level was reported by 75% of the families. Two children were born to consanguineous parents. A familial cancer history was noted in 41.7% of patients. Clinical symptoms were dominated by headache (83.3%) and vomiting (66.7%). Regarding treatment, two patients were treated with surgery and seven with a combination of surgery, radiotherapy, and chemotherapy. During the period of study, there were no cases of death. A good outcome was noted in 91.7% of patients. Only one child received palliative care. Childhood cancer in general, and medulloblastoma in particular, is on the rise in Morocco. Given the scarcity of publications on this type of cancer in our country, a national registry is necessary for the monitoring and appropriate management of this disease.
MDB-11. AN ONLINE CALCULATOR USING MACHINE LEARNING FOR PREDICTION OF SURVIVAL IN PEDIATRIC PATIENTS WITH MEDULLOBLASTOMA
Authors: Kuo Cathleen , Brown Nolan , Lim Jaims , Monteiro Andre , Recker Matthew , Ghannam Moleca M , Gendreau Julian , Li Veetai , Reynolds Renée
Published: June 2023

OBJECTIVE

Medulloblastoma is the most common malignant intracranial tumor affecting the pediatric population. Despite advancements in multimodal treatment over the past 2 decades yielding a >75% 5-year survival rate, children who survive often have substantial neurological and cognitive sequelae. The authors aimed to identify risk factors and develop a clinically friendly online calculator for prognostic estimation in pediatric medulloblastoma patients.

METHODS

Pediatric patients with a histopathologically confirmed medulloblastoma were extracted from the Surveillance Epidemiology and End Results database (2000-2019) and split into training and validation cohorts in an 80:20 ratio. The Cox proportional hazards model was used to identify the univariate and multivariate survival predictors. Subsequently, a calculator with those factors was developed to predict 2-, 5-, and 10-year overall survival as well as median survival months for pediatric medulloblastoma patients. The performance of the calculator was determined by discrimination, calibration, and decision curve analysis (DCA).

RESULTS

A total of 1,739 pediatric patients with medulloblastoma met the prespecified inclusion criteria. Fourteen variables, including age, sex, race, ethnicity, median household income, county attribute, laterality, histology, anatomical location, tumor grade, tumor size, surgery status, radiotherapy, and chemotherapy, were included in the calculator (https://spine.shinyapps.io/Peds_medullo/). The concordance index was 0.757 in the training cohort and 0.762 in the validation cohort, denoting clinically useful predictive accuracy. Good agreement between the predicted and observed outcomes was demonstrated by the calibration plots. The DCA curves indicated that the developed model has a good clinical prognostic benefit for pediatric medulloblastoma patients.

CONCLUSION

An easy-to-use prognostic calculator for a large cohort of pediatric patients with medulloblastoma was established. Future efforts should focus on improving granularity of population-based registries and externally validating the proposed calculator.
MDB-12. TRANS-SPECIES ANALYSIS OF REPLICATION-REPAIR DEFICIENT (RRD) MEDULLOBLASTOMA AND RESPONSE TO IMMUNE-CHECKPOINT INHIBITION: AN IRRDC REPORT
Authors: Das Anirban , Fernandez Nicholas R , Levine Adrian , Smith Kyle , Wang Evan , Galati Melissa , Aamir Zoya , Chung Jill , Negm Logine , Crump Owen , Trinh Quang , Nunes Nuno M , Bianchi Vanessa , Stengs Lucie , Edwards Melissa , Stein Lincoln , Bouffet Eric , Taylor Michael , Northcott Paul , Ramaswamy Vijay , Hawkins Cynthia , Tabori Uri
Published: June 2023

BACKGROUND

Replication-repair deficiency (RRD) stemming from mismatch repair and polymerase-proofreading gene defects (MMRD/PPD) lead to hypermutant childhood cancers, most frequently brain tumors. While medulloblastoma is reported, the clinical, genomic and immune landscape remains unknown.

METHODS

We analysed the genome, methylome, transcriptome (bulk, single-nuclei) and immune-microenvironment of the largest cohort of RRD-medulloblastoma patients enrolled by the International RRD Consortium, and correlated these to clinical outcomes. RRD mice-models were used to preclinically assess response to immunotherapy.

RESULTS

RRD-medulloblastoma (n=40) were enriched for anaplasia (55%) and localised disease (85%). Methylation/nano-string-based subgrouping failed to classify 40%, while the remaining clustered with the SHH-subgroup. Copy-number changes were notably infrequent (<20%). All tumors harboured hypermutation and microsatellite instability in contrast to non-RRD controls (p<0.0001). Pathogenic variants were frequent in POLE/POLD1 (80%), TP53 (48%) and SHH-pathway genes (PTCH1, SUFU, SMO: 56%). Interestingly, some tumors additionally had glioma-driver alterations (ATRX, NF1: ~50%), similar to RRD cerebellar glioblastoma, but distinct from both non-RRD medulloblastoma (n=791) and hemispheric glioblastoma (n=733) controls. Moreover, although bulk-transcriptome matched non-RRD SHH-medulloblastoma, single-nuclei analyses suggested an earlier cell-of-origin potentially explaining the heterogenous phenotypes resulting from the driver mutations. Uniquely, immune analyses (gene expression and immunohistochemistry) demonstrated high intra-tumoral CD8 T-cell infiltration.

Three-year progression-free survival was 60%. Outcome was worse for RRD-medulloblastoma harbouring TP53-mutation (p=0.04) and failing subgroup-classification (p=0.004). Nestin/Cre MSH2-/- POLE;p.S459F mice developing medulloblastoma recapitulated human disease and demonstrated response to anti-PD1 monotherapy. Recurrent/progressive human tumors including those harbouring TP53-mutations exhibited radiological responses to anti-PD1 monotherapy, which was associated with prolonged ongoing survival in comparison to those not treated with checkpoint-inhibitors (p=0.02).

CONCLUSIONS

Hypermutant RRD-medulloblastoma reveal heterogenous phenotypes due to unique spectrum of driver mutations and an early cell of origin. Their immune-hot microenvironment allows successful salvage treatment with checkpoint-inhibitors for those failing chemo-radiation, including the high-risk SHH/TP53-mutant and unclassified subgroups.
MDB-14. MULTI-OMICS OF HUMAN MEDULLOBLASTOMAS AND GENETIC LINEAGE TRACING IN VIVO IDENTIFY REGULATORS OF STEM-CELL MAINTENANCE
Authors: Yu Bohyeon , Jung Jangham , Hoare Owen , Shamardani Karin , Diaz Aaron
Published: June 2023

Recent studies implicate the glutamatergic lineage of the rhombic lip (RL) as the origin of group 3/4 medulloblastomas (MBs). MB stem cells resemble stalled RL progenitors and targeting common pluripotency factors induces differentiated phenotypes. However, the genetic and epigenetic regulators of stemness in MB remain incompletely understood. We profiled tissues resected from 38 patients who underwent surgery for MB at UCSF via single-nucleus RNA sequencing (snRNA-seq). Of these, 13 were also subjected to single-cell assay for transposase-accessible chromatin by sequencing (scATAC-seq). These assays generated 560,509 and 71,276 cells respectively, after quality control. Motif and latent-space analyses were used to infer cell-type specific transcription-factor expression and binding, and associated cis-regulatory enhancers. Further, we performed single-cell genetic lineage tracing on xenografts obtained from two distinct group-3/4 patient-derived lines, engineered to express heritable barcodes. We trained a machine-learning classifier on public snRNA-seq of human RL and classified Nestin-high quiescent stem-like cells, Nestin-moderate/ASPM-high proliferating progenitor cells, and glutamatergic lineage cells at various stages of differentiation in our MB clinical specimens and xenografts. Integrated analysis of these datasets maps the transition from quiescent to proliferating stem-like MB cells to more differentiated cell types at the level of transcription-factor activity and binding. We found that both the expression and binding of epigenetic regulators SUZ12, EZH2, DNMT1, and BRD7/8 were high in stem cells, but attenuated during lineage commitment. This was also the case for genes known to maintain neural stem cells in the developing brain, e.g. LIN28, FOXO3, HES6, LHX1/2/4, NUP98. Comparative analysis of developmental and MB gene expression, with motif analysis, implicates a conserved embryonal mechanism for indefinite replicative division in MB stem-like cells. These studies provide a single-cell atlas of transcriptomic and epigenetic cellular states in human MB, a map of MB cellular hierarchical organization and transcription-factor regulation of stem-cell maintenance in vivo.
MDB-15. CLINICAL CHARACTERISTICS AND OUTCOME OF CHILDHOOD LI-FRAUMENI SYNDROME MEDULLOBLASTOMA PATIENTS
Authors: Kolodziejczak Anna , Guerrini-Rousseau Lea , Planchon Julien Masliah , Mynarek Martin , Sill Martin , Autry Robert , Zhao Eric , Hirsch Steffen , Dufour Christelle , Ayrault Olivier , Waszak Sebastian , Ramaswamy Vijay , Pentikainen Virve , Clifford Steven , Schwalbe Ed , Massimi Luca , Snuderl Matija , Karajannis Matthias , White Christine , Redmond Shelagh , Loizos Loizou , Jakob Marcus , Schmid Irene , Hauer Julia , Blattmann Claudia , Filippidou Maria , Sutter Christian , Pietsch Torsten , Frank Stephan , Schewe Denis , Malkin David , Sehested Astrid , Wong Tai-Tong , Carceller Fernando , Mueller Sabine , Taylor Michael , Tabori Uri , Bouffet Eric , Kool Marcel , Sahm Felix , Kratz Christian , Sturm Dominik , Jones David , Rutkowski Stefan , Witt Olaf , Bougeard Gaëlle , Pajtler Kristian , Pfister Stefan , Bourdeaut Franck , Milde Till
Published: June 2023

Patients with Li-Fraumeni syndrome (LFS) who develop medulloblastoma (MB) have a very poor prognosis. The development of novel therapeutic strategies is challenged by the lack of clinical data for this patient group. We here present clinical and molecular data on a retrospective cohort of pediatric patients with LFS-associated MB.

This is an international, retrospective, multicenter cohort study. Patients with LFS-associated MB under 21 years and class 5 (pathogenic) or class 4 (likely pathogenic) constitutional TP53 variants were included. We evaluated TP53 mutation status (constitutional and somatic), DNA methylation subgroup, treatment modalities, event-free (EFS) and overall survival (OS), patterns of recurrence, as well as occurrence of secondary neoplasms.

Forty-seven individuals with LFS-associated MB were included. MBs were classified mainly as Sonic Hedgehog (SHH) group (87%). TP53 variants were classified as class 5 (70%) and class 4 (30%). The majority (74%) of TP53 variants represented missense variants. The 2-year (y-) EFS and -OS were 33% and 53%, respectively. A significantly better outcome was seen in patients who received post-operative radiotherapy (RT) (2y-EFS: 44%, 2y-OS: 60%) or chemotherapy before RT (2y-EFS: 24%, 2y-OS: 48%) compared with patients who received no RT (2y-EFS: n.a., 2y-OS: 25%). No significantly different outcomes were seen between patients treated either according to protocols including high-intensity chemotherapy or receiving only maintenance-type chemotherapy (2y-EFS: 42% and 31%, 2y-OS: 68% and 53%, respectively).

Patients with LFS-associated MB have a dismal prognosis. Use of RT in LFS-associated MB significantly increased survival rates in the presented cohort, but choice of chemotherapy regimen did not influence their clinical outcome. To improve the outcome of patients with LFS-associated MB, prospective collection of clinical data and development of novel treatments are required.
MDB-20. IMPROVING THE PERSISTENCE OF CAR T CELLS AGAINST GROUP 3 MEDULLOBLASTOMAS VIA EPIGENETIC REGULATION OF EXHAUSTION
Authors: Dukes Meghan , Houke Haley , Zindy Frederique , Youngblood Ben , Roussel Martine , Krenciute Giedre
Published: June 2023

Medulloblastomas (MB) are the most common malignant pediatric brain tumor with molecular subclass Group 3 (G3) requiring the most intense treatment regimen including surgery, intensive radiation followed by chemotherapy. Despite an overall 40% survival rate, survivors’ quality of life is often impaired due to whole-brain radiation leading to neurocognitive defects. Chimeric antigen receptor (CAR) T cell therapy has emerged clinically as a safe, tumor-targeted immunotherapy with the most success observed against hematological malignancies. Translating CAR T cell therapy to brain tumors has proven challenging due to lack of targetable antigens on the surface of tumor cells and lack of persistence of CAR T cells. We have previously identified that patient samples and PDOX-derived cell lines of G3 MBs consistently over-express B7-H3 (CD267) compared to normal tissue. Herein, we investigated B7-H3 targeted CAR T cell products against G3 MB in vitro and in vivo. Initial results show that B7-H3 CAR T cells are highly effective in clearing human MB tumors in NSG mice, however, 4 out 5 mice experienced late relapses that showed reduced, but still present, B7-H3 antigen expression. We hypothesize that the lack of CAR T cell persistence is ultimately responsible for tumor regrowth. To overcome this limitation, we have identified epigenetic regulators DNMT3A and TET2 that control T cell exhaustion. Using CRISPR/Cas9, we genetically knocked out DNMT3A and TET2 in CAR T cells and investigated the impact on persistence and anti-tumor activity. Our data supports the hypothesis that knock out of DNMT3A and TET2 improves antigen-dependent proliferation and persistence of B7-H3 CAR T cells against G3 MB tumor lines in vitro. DNMT3A KO shows a more sustained effect over TET2. Currently, we are optimizing in vivo experiments to investigate prolonged tumor clearance in both immunocompromised and fully immunocompetent murine models.
MDB-21. THE OTX2-REGULATED ALTERNATIVE SPLICING LANDSCAPE CONTROLS DIFFERENTIATION AND RECONSTITUTES THE DEVELOPMENTAL ORIGINS OF GROUP 3 MEDULLOBLASTOMA
Authors: Saulnier Olivier , Zagozewski Jamie , Liang Lisa , Hendrikse Liam D , Gordon Victor , Aldinger Kimberly A , Haldipur Parthiv , Borlase Stephanie , Coudiere-Morrison Ludivine , Layug Paul , Cai Ting , Porter Christopher J , Richard Stephane , Doble Brad W , Millen Kathleen J , Taylor Michael D , Werbowetski-Ogilvie Tamra E
Published: June 2023

Medulloblastoma (MB) is the most common malignant primary pediatric brain tumor with Group 3 MB exhibiting the worst prognosis. Overexpression/amplification of OTX2 is a molecular hallmark of Group 3 MB and is primarily known to control tumor growth through regulation of cell cycle. Here, we utilized mass spectrometry and RNA-sequencing to report a non-canonical role for OTX2 in regulating alternative splicing. RNA-sequencing conducted on two OTX2-silenced Group 3 MB tumorsphere models identified 48 differentially spliced genes (DSG) associated with RNA processing, splicing and neurodevelopment that were common between both cell lines. Further interrogation of the OTX2-regulated DSGs revealed that 15 are also significantly correlated with OTX2 expression in a set of Group 3/Group 4 MB patient samples. Pseudotime mapping of the developmental trajectories of the DSGs onto the developing rhombic lip (RL) revealed significant overlap with RL lineages, including the majority of the 15 shared OTX2-regulated DSGs. We then functionally validated specific alternatively spliced exons of two DSGs, PPHLN1 and MADD which represent “early” and “late” RL developmental stages, respectively. PPHLN1 and MADD splice-blocking morpholinos were utilized to drive exon skipping and recapitulate splicing events identified in OTX2-silenced or OTX2-wildtype tumorspheres. Exon skipping altered tumorigenic properties of Group 3 MB cells in vitro, including tumorsphere size and number. Additionally, intracerebellar injections of Group 3 MB cells treated with PPHLN1 morpholino impaired tumor initiation and growth in vivo. Further breakdown of Group 3/Group 4 MBs by PPHLN1 and MADD percent spliced in (PSI) revealed that the PPHLN1 high/MADD low PSI signature observed in our OTX2 wildtype tumorspheres is associated with the most aggressive and primitive Group 3g subtype. Together, our findings reveal a previously unrecognized regulatory role for OTX2 in controlling Group 3 MB AS and offer fresh opportunities to exploit MB-specific AS events therapeutically to abrogate tumor progression.
MDB-23. ELP1 GERMLINE DEFICIENCY SENSITIZES THE GRANULE NEURON LINEAGE TO SHH MEDULLOBLASTOMA AND EXPOSES NOVEL THERAPEUTIC VULNERABILITIES
Authors: Garcia-Lopez Jesus , Ahmad Shiekh Tanveer , Li Yiran , Gudenas Brian , Kojic Marija , Manz Friedrik , Jonchere Barbara , Mayasundari Anand , Pitre Aaron , Hadley Jennifer , Paul Leena , Batts Melissa , Pfister Stefan , Waszak Sebastian , Bianski Brandon , Tinkle Christopher , Orr Brent , Rankovic Zoran , Robinson Giles , Wainwright Brandon , Kutscher Lena , Lin Hong , Northcott Paul
Published: June 2023

Germline loss-of-function (LOF) variants in Elongator complex protein 1 (ELP1) are the most prevalent predisposing genetic events in childhood medulloblastoma (MB). ELP1 germline carriers develop SHH-MBs that exhibit coincident somatic PTCH1 mutations and universal loss-of-heterozygosity of the remaining ELP1 allele through chromosome 9q deletion. The molecular, biochemical, and cellular mechanisms by which germline ELP1/Elongator deficiency contribute to SHH-MB tumorigenesis remain largely unknown. Herein, we report that mice engineered to mimic germline Elp1 LOF (i.e., Elp1HET) seen in SHH-MB patients exhibit hallmark features of premalignancy events in cycling cerebellar granule neuron progenitors (GNPs), the lineage-of-origin for SHH-MB. Compared to wild-type counterparts, Elp1HET GNPs exhibit increased replication stress-associated DNA damage, homologous recombination-associated genomic instability, accelerated cell cycle kinetics, reduced p53-dependent apoptosis in response to genotoxic stress, and slowed differentiation. Orthotopic transplantation of Elp1HET GNPs harboring somatic Ptch1 inactivation into the cerebella of immunocompromised mice promotes onset of SHH-MB tumors with incomplete penetrance that exhibit reduced p53 transcriptional activity through a currently unknown mechanism(s). Concomitant Elp1 and Ptch1 gene targeting in p53-null GNPs reproduces highly penetrant cerebellar tumors recapitulating the molecular and phenotypic features of ELP1-associated SHH-MB. Finally, reactivation of the p53 pathway through preclinical treatment with an MDM2 inhibitor promotes cell death and prolongs the survival of patient-derived xenograft tumor (PDX) models harboring deleterious ELP1 mutations. Together, our findings reveal that germline Elp1 LOF heightens genomic instability and malignant transformation in cycling GNPs, providing a mechanistic model for the subgroup-restricted pattern of predisposition associated with pathogenic ELP1 germline carriers. These results provide essential mechanistic insight into the molecular and cellular basis of SHH-MB predisposition driven by ELP1 LOF and nominate therapies that overcome p53 pathway inhibition as a rational treatment option for affected children.
MDB-29. MEDULLOBLASTOMA SUBGROUP-SPECIFIC CHROMATIN STATES REVEAL NOVEL THERAPEUTIC VULNERABILITIES
Authors: Tao Ran , Gao Qingsong , Erkek Serap , Matsui Yurika , Djekidel Nadhir , Darrow Emily , Kumar Rahul , Smith Kyle , Wu Gang , Korbel Jan , Peng Jamy , Pfister Stefan , Northcott Paul
Published: June 2023

Medulloblastoma (MB) is a highly malignant childhood brain tumor comprising a collection of molecularly and clinically distinct entities. Mutations in chromatin-modifying genes distribute across subgroups and account for nearly half of all MBs. However, the molecular consequences of chromatin modifier alterations remain poorly understood. Herein, we characterize the chromatin landscape of fifty-two primary MBs representing each MB subgroup using chromatin immunoprecipitation followed by sequencing (ChIP-seq) for six histone marks and perform multi-layered integration with somatic genetics, genome-wide DNA methylation, and transcriptomic datasets extracted from the same tumors. Analysis of differential chromatin states across MB subgroups identified significant enrichment of a bivalent enhancer state (EnhBiv) in Group 4 MB. Group 4-specific EnhBivs mapped to promoters of key neurodevelopmental genes and transcription factors and was associated with increased DNA methylation and reduced expression. In accordance with higher EnhBivs coverage in Group 4 MB, we found that KDM2B, a H3K4me3 and H3K36me2 demethylase, is highly expressed in Group 4 MBs and patient-derived xenografts (PDXs). KDM2B knockout mediated by CRISPR targeting suppressed growth of both Group 3 MB cell lines in vitro and Group 4 MB PDXs in vivo but had no effect on control SHH-MB cells, suggesting that KDM2B is a novel selective dependency in MB. Knockout of KDM2B in MB cells induced profound upregulation of PRC2 targets, genes regulated by bivalent histone marks, as well as genes involved in neuronal differentiation. Further biochemical and molecular studies demonstrated that KDM2B interacts with PRC2 complex subunits and occupies gene promoters associated with Group 4-specific EnhBiv chromatin. In summary, our comprehensive characterization of the MB chromatin landscape in a large cohort of primary tumors provides novel insights into the epigenetic basis of MB and implicates KDM2B as a novel Group 4 MB dependency that should be prioritized as a therapeutic target in this subgroup.
MDB-30. IDENTIFICATION AND CHARACTERIZATION OF NOVEL TRANSCRIBED ULTRACONSERVED REGIONS AS LONG NON-CODING RNAS IN MEDULLOBLASTOMA
Authors: Hudson Kadie , Gibert Jr. Myron K , Zhang Ying , Dube Collin , Saha Shekhar , Vernon Ashley , Abounader Roger
Published: June 2023

Medulloblastoma is the most common pediatric brain tumor. While the 5-year survival rate for medulloblastoma patients has improved with molecular subgrouping, children who undergo standard of care treatments frequently experience permanent neurological deficits. Additionally, under-characterized medulloblastoma subgroups lack targeted therapies, correlating to lower survival rates for these patients. To find new therapies, a better understanding of the molecular basis of medulloblastoma is needed. This work investigates the role of “Transcribed Ultra-Conserved Regions” (TUCRs) in medulloblastoma about which there is no published literature. TUCRs represent a distinct group of 481 putative lncRNAs that are 100% conserved across human, mouse, and rat genomes. These ultra-conserved elements change 20 times slower than the average genomic region and are therefore under intense negative selection within multiple species. Since their discovery, TUCRs have only been correlated to a handful of cancers and remain exceedingly underexamined within cancer research. Using RNA-sequencing and differential gene expression analysis of medulloblastoma tumors as compared to normal human cerebellum samples, we identified a multitude of differentially expressed TUCRs spanning across all TUCR genomic annotations. In vitro experimentation using several medulloblastoma established cell lines indicated significant upregulation of these TUCR transcripts. We are currently investigating the functional effects and mechanisms of action of select differentially expressed TUCRs in medulloblastoma. Altogether, our work is investigating the oncogenic and tumor suppressive nature of TUCRs. It represents the first study of TUCRs in medulloblastoma that will significantly advance our basic understanding of this highly understudied class of molecules.
MDB-34. “INDUCING IRON IMBALANCE BY TARGETING THE ABCB7/GPX4 AXIS ACCELERATES FERROPTOSIS IN MEDULLOBLASTOMA”
Authors: Kanchan Ranjana , Perumal Naveenkumar , Doss David , Khan Parvez , Venkata Ramakanth , Thapa Ishwor , Vengoji Raghupathy , Kaushal Jyoti , Siddiqui Jawed , Nasser Mohd Wasim , Batra Surinder , Mahapatra Sidharth
Published: June 2023

Medulloblastomas (MB), the most common malignant pediatric brain tumor and a leading cause of childhood mortality, are stratified into four primary subgroups. Deletions within chromosomal locus 17p13.3, which houses multiple tumor suppressor genes including miR-1253, characterize high-risk group 3 tumors. These aggressive tumors also enrich iron transport genes to satisfy their high proliferative need. MiR-1253 targets iron transport by inhibiting the mitochondrial Fe-S transporter, ABCB7. This study elucidated the impact of repressing ABCB7 on cisplatin cytotoxicity in group 3 MB and whether these effects were mediated by ferroptosis. In silico and in vitro analyses revealed specific enrichment of ABCB7 and GPX4, a critical regulator of ferroptosis, in group 3 MB cell lines and tumors. MiR-1253 overexpression (miR-1253OE) resulted in downregulation of both ABCB7 and GPX4, concurrently increasing mitochondrial iron overload, mitochondrial oxidative stress, and lipid peroxidation, leading to cell death and abrogation of medullosphere formation; ABCB7 knockdown (ABCB7KD) recapitulated these effects and abrogated GPX4 expression. Fractionation studies confirmed the inhibitory impact of miR-1253OE and ABCB7KD on GPX4 expression in the cytosol and mitochondria. Seahorse studies revealed that the bulk of ATP generation was occurring in the cytoplasm through glycolysis and not via oxidative phosphorylation, suggesting mitochondrial dysfunction was triggered when ABCB7 was repressed. Cisplatin, a chemotherapeutic agent used in group 3 MB treatment, induces cell death by DNA crosslinking; it also inhibits GPX4 expression. In miR-1253OE and ABCB7KD group 3 cancer cells, cisplatin IC50 was reduced 2-fold. Resultantly, cisplatin treatment augmented oxidative stress and lipid peroxidation, depleted glutathione stores, and culminated in a higher index of ferroptosis. In a mouse model of group 3 tumors, ABCB7KD potentiated cisplatin effects and dramatically prolonged survival. The current study illustrates how targeting iron transport can augment ferroptosis to potentiate cisplatin cytotoxicity in group 3 MB tumors.
MDB-35. RECURRENT MEDULLOBLASTOMA A RARE COMPLETE RESPONSE IN EXTRA NEURAL DISEASE
Authors: Schwartz Jonathan , Weinmann Dena , Johnson Heather , Soumen Khatua , Neth Bryan , Keating Gesina
Published: June 2023

Extra-neural metastasis in recurrent medulloblastoma, carries a potentially fatal outcome. No robust salvageable therapeutic strategy is present, for these clinical phenotypes. We present an 18-year male, who presented at the age of 8 years as a standard risk medulloblastoma and was treated with chemotherapy (as per ACNS0331) followed by craniospinal radiation. At first recurrence in the thoracic spine, he underwent a gross total resection of the tumor, followed by high-dose stem cell chemotherapy and focal radiation. The 2nd recurrence which occurred in the thecal sac 9 years later was treated with metronomic anti angiogenic chemotherapy with outcome a complete radiographic response. Third recurrence occurred in the liver, bone marrow, multiple bones of axial skeleton and sacrum. Molecular interrogation of the lesions showed a (non-WNT / non-SHH subtype) with absence of MYC or MYCN amplification or any other clinically significant biological aberration. He received local radiation to tumors, following which he developed severe myelosuppression. A bone marrow aspirate and biopsy showed greater than 95% involvement with medulloblastoma cells. He was treated with irinotecan and temozolomide. A dramatic response was noted after one cycle (with PET and MRI), with bone marrow involvement of less than 1%. He continues with that therapy. To-date no defined molecular profile is predictive of extra neural metastasis, and has been likened to a fatal outcome, most patients being declared for hospice care. Our patient with this unique clinical outcome, warrants extensive evaluation of these rare clinical phenotypes including methylation profiling and RNA sequencing, which might suggest, certain molecular aberrations which correlates with good response, even in these biologically aggressive variants.
MDB-38. COMPUTATIONAL DRUG SENSITIVITY PREDICTS MEDULLOBLASTOMA SUBGROUP-SPECIFIC THERAPEUTICS
Authors: Jermakowicz Anna , Suter Robert , Ruiz Luz , Ayad Nagi
Published: June 2023

Medulloblastoma is the most common malignant pediatric brain tumor. Tumors are typically characterized as Group 3, Group 4, SHH, or WNT. Current standard-of-care includes surgery, radiation, and chemotherapy; however, treatment response and prognosis vary widely between subgroups. Additionally, surviving children frequently suffer from lifelong neurocognitive deficits. Therefore, novel therapeutic options are urgently needed. Despite extensive characterization of medulloblastoma tumors into four molecular subgroups, few subgroup specific therapies have advanced to the clinic. To address this, we have developed a novel platform called DrugSeq, which predicts drug sensitivities in patients and can stratify tumors by subgroup. DrugSeq also identifies key pharmacotranscriptomic differences between primary and recurrent tumors. We first calculated disease signatures for each patient by normalizing gene expression to low grade glioma samples from the posterior fossa. Using previously developed transcriptional consensus signatures (TCSs) that represent the affect that a drug has on the gene expression across a panel of cancer cell lines, we then calculated the discordance between each patient disease signature and drug TCS. Finally, using an ANOVA analysis we identified drugs which are predicted to differentially target each medulloblastoma subgroup. Among our top predicted anti-cancer compounds we found several kinase inhibitors, bromodomain inhibitors, and several psychiatric drugs with known brain penetrance. Additionally, we show distinct differences in drug sensitivity predictions between newly diagnosed and recurrent tumors, such as sensitivity to BET inhibition for recurrent Group 3 and Group 4 tumors despite no predicted sensitivity in the newly diagnosed tumors. Future studies with larger datasets may be able to further subdivide patients by age or molecular features within subgroups. Collectively, we show that DrugSeq may identify novel therapies and facilitate patient stratification in clinical trials, leading to more successful targeted medulloblastoma therapies.