MB Patients Research Articles

Treatment response as surrogate to predict risk for disease progression in pediatric medulloblastoma with persistent magnetic resonance imaging lesions after first-line treatment.

This study aims at clarifying the impact of persistent residual lesions following first-line treatment for pediatric medulloblastoma. Data on 84 pediatric patients with medulloblastoma and persistent residual lesions on centrally reviewed magnetic resonance imaging (MRI) at the end of first-line therapy were analyzed. Twenty patients (23.8%) had residual lesions in the tumor bed (R+/M0), 51 (60.7%) had distant lesions (R0/M+) and 13 (15.5%) had both (R+/M+). Overall response to first-line therapy was minor or partial (≥ 25% reduction, minor response [MR]/PR) for 64 (76.2%) and stable disease (SD) for 20 patients (23.8%). Five-year post-primary-treatment progression-free (pptPFS) and overall survival (pptOS) were superior after MR/PR (pptPFS: 62.5 ± 7.0%[MR/PR] vs. 35.9 ± 12.8%[SD], P = .03; pptOS: 79.7 ± 5.9[MR/PR] vs. 55.5 ± 13.9[SD], P = .04). Furthermore, R+/M + was associated with a higher risk for progression (5-year pptPFS: 22.9 ± 17.9%[R+, M+] vs. 72.4 ± 12.0%[R+, M0]; P = .03). Watch-and-wait was pursued in 58 patients, while n = 26 received additional treatments (chemotherapy only, n = 19; surgery only, n = 2; combined, n = 3; valproic acid, n = 2), and their outcomes were not superior to watch-and-wait (5-year pptPFS: 58.5 ± 7.7% vs. 51.6 ± 10.7% P = .71; 5-year pptOS: 76.3 ± 6.9% vs. 69.8 ± 9.7%, P = .74). For the whole cohort, 5-year pptPFS by molecular subgroup (58 cases) were WNT: 100%, SHH: 50.0 ± 35.4%, group-4, 52.5 ± 10.5, group-3 54.2 ± 13.8%; (P = .08). Overall response and extent of lesions can function as surrogate parameters to predict outcomes in pediatric MB patients with persistent lesions after first-line therapy. Especially in the case of solitary persistent medulloblastoma MRI lesions, additional therapy was not beneficial. Therefore, treatment response, extent/kind of residual lesions and further diagnostic information need consideration for indication of additional treatments for persisting lesions.

Abstract 3346: JMJD6 as a novel tumorigenic factor and therapeutic target in group 3 (MYC-driven) medulloblastoma

Abstract Medulloblastoma (MB) is the most common type of childhood brain cancer worldwide. Although current treatment with surgery and extensive chemoradiation has led to increased survival rates, many MB patients still die from the disease. Moreover, surviving patients suffer severe long-term side effects as a consequence of treatment. It is therefore crucial to develop more effective and less toxic therapies. The most aggressive subtype of MB tumors often exhibits amplification or overexpression of the MYC oncogene. Patients with MYC-amplified MB exhibit a high frequency of cerebrospinal tumor dissemination, often experience treatment resistance and have extremely poor prognoses. While the MYC oncogene is established as the oncogenic driver in Group 3 MB, it has remained undruggable. Thus, targeting regulatory components of MYC and the signaling pathways regulated by it is of great potential therapeutic value. Studies have revealed that MB has very few germline mutations in cancer predisposition genes, suggesting that dysregulated epigenetic pathways might be critical in MB pathogenesis. Particularly, dysregulation of epigenetic modifiers, including histone methyltransferases and histone demethylases, is very common in Group 3 MB, compared to other MB subgroups. Therefore, it is important to identify such epigenetic modifiers that may have controls on MYC and its tumorigenic activities and explore these as the epigenetic drug-candidate targets in Group 3 (MYC-driven) MB. In this regard, we found that protein arginine demethylase Jumonji C domain-containing protein 6 (JMJD6), an emerging key epigenetic enzyme in cancers, is a novel regulator of MYC expression in MYC-driven MB. We observed high levels of JMJD6 that not only mirror MYC expression in the most aggressive MB but also correlate with poor outcomes in these patients. Knockdown of JMJD6 decreased MYC expression, cellular proliferation/survival and stemness in MYC-amplified MB cells. Mechanistically, our results revealed that JMJD6 forms complexes with proteins involved in maintaining and regulating the promoter-pause at super-enhancers, suggesting that JMJD6 can regulate MYC at the transcription level. Moreover, our in vivo analyses of JMJD6 inhibition, either with inducible gene knockdown or a pharmacologic small molecule inhibitor, demonstrated anti-MB potential with suppressed MYC expression. Based on this background and preliminary observations, we hypothesize that JMJD6 plays crucial roles in the most aggressive MB by regulating MYC expression and hence MYC-driven tumorigenesis. Accordingly, we hypothesize that targeting the JMJD6-MYC axis by JMJD6 inhibition can serve as a powerful therapeutic strategy for MYC-driven MB. Citation Format: Matthew Kling, Devendra Kumar, Sutapa Ray, Shantaram Joshi, Don Coulter, Nagendra K. Chaturvedi. JMJD6 as a novel tumorigenic factor and therapeutic target in group 3 (MYC-driven) medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3346.

Abstract 5107: Targeting CTPS1 and nucleotide biosynthesis pathways as a novel therapeutic approach in MYC-amplified medulloblastoma

Abstract Although leukemia is the leading pediatric oncology diagnosis, brain tumors are the leading cause of pediatric cancer-related death. Medulloblastoma is the most common pediatric brain tumor, representing approximately 20% of all diagnoses. Of all four defined subgroups, MYC-amplified Group 3 medulloblastoma (G3 MB) tumors are the deadliest, with a < 50% 5-year overall survival rate. High risk patients receive large doses of chemo- and radio- therapy (CT/RT) with most survivors experiencing debilitating neurological and cognitive sequelae making the identification of novel targets of the utmost importance. Utilizing whole-genome CRISPR screening from the Pediatric Cancer Dependency Map, we identified the enzyme CTPS1 as a strong G3 MB-specific vulnerability. Recent studies corroborate our findings and suggest a dependence on the de novo pyrimidine biosynthesis pathway in MB. Targeted knockdown of CTPS1 restricts sphere formation and reduces proliferation of G3 MB tumor lines. We confirmed this genetic approach using a specific CTPS1 inhibitor, which also reduced proliferation but did not induce cell death, suggesting a cytostatic mechanism. Strikingly, we demonstrate that G3 MB is only dependent upon de novo pyrimidine synthesis and not de novo purine synthesis, despite regulation of both pathways by oncogenic MYC signaling. Previous work has shown that triggering an imbalance in pyrimidine and purine levels leads to replication stress and activation of the ATR/CHK1 pathway. Both genetic and pharmacologic inhibition of CTPS1 leads to CHK phosphorylation, allowing for cell survival despite lack of dNTPs. Previously, the CHK1 inhibitor, prexasertib, demonstrated robust brain accumulation and targeting of G3 MB in pre-clinical models. Dual inhibition of CHK1 and CTPS1 synergizes to significantly reduce cell viability in vitro and tumor growth in vivo. We further demonstrated robust synergy of CHK1 inhibition with either glutamine antagonists or other inhibitors of the de novo pyrimidine synthesis pathway, prolonging in vivo survival in an orthotopic xenograft G3 MB model. The results of this investigation confirm CTPS1 as a novel target in G3 MB, pinpoint a dependency on de novo pyrimidine rather than purine biosynthesis, and identifies a novel rational combination for treatment in MYC-amplified MB patients. Citation Format: Matthew Hathaway, Hawa Jagana, Michael Meechan, John Hemenway, Kate E. Gadek, Aya Miyaki, Isabella Terrones, Barbara Slusher, Siobhan Pattwell, Nicholas Vitanza, Myron K. Evans. Targeting CTPS1 and nucleotide biosynthesis pathways as a novel therapeutic approach in MYC-amplified medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5107.

Abstract 5112: Discovery of nuclear envelope mislocalization in MYC-driven group 3 medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor, comprised of four distinct molecular subgroups (WNT, SHH, Group 3, and Group 4). A subset of Group 3 MB tumors harbor focal amplifications of the MYC oncogene and are particularly prone to tumor recurrence and leptomeningeal spread which currently remains incurable. Therefore, there is an urgent need for the development of therapeutic modalities that can spare the vulnerable, developing brain while having potent antitumor efficacy against recurrent MB. Through proteomic profiling of cyclophosphamide-resistant patient-derived MYC-driven Group 3 MB cell line SU_MB002, we identified nuclear envelope (NE) proteins to be highly abundant and enriched in Group 3 MB compared to normal tissue. We discovered that LBR, an inner nuclear transmembrane protein, while present in all cells at the NE, is aberrantly presented to the cell surface in MYC-driven Group 3 MB cell lines. Immunohistochemistry analysis of LBR on MB patient tumor samples identified that positive LBR staining correlated with a significantly worse prognosis and was also identified to be enriched in recurrent tissue compared to their matched primary samples. Cell surface expression flow cytometry analysis identified highly abundant cell surface expression in all MYC-driven Group 3 MB cell lines with low to no expression in human neural stem cells and other MB subgroups. Mechanistically, high resolution microscopy of endogenously HALO-tagged LBR reveals LBR cell surface presentation to be linked to ER or ER-like vesicles that are directly trafficked to the cell surface. Transcriptomic analysis of LBR cell surface positive and negative cells identify that cell surface positivity is significantly linked to cell division and mitotic processes (cell cycle process, mitotic cell cycle process, chromosome segregation, cell division, nuclear division). LBR mislocalization stems from mitosis at the time of nuclear envelope dissolution and is enriched in the hyperproliferative, therapy-resistant subpopulation of MB cells following exposure to chemotherapy and radiation in vitro and in vivo and may act as a marker for the brain tumor initiating cells that seed recurrence. Therefore, we demonstrate that LBR is an ideal therapeutic target for patients facing relapsed or treatment-refractory MB and have generated single domain antibodies in an effort to engineer LBR-specific CAR T cells. The development of therapies against NE proteins like LBR may provide patients a novel therapeutic opportunity that may be able to treat therapy-resistant, hyperproliferative MYC-driven Group 3 MB cells. Citation Format: Yujin Suk, Jorge Ibañez-Vega, Iqra Chaudhry, Martin Rossotti, Minomi Subapanditha, Petar Miletic, Stefan Custers, Laura Escudero, Alberto Delaidelli, Takuma Nakashima, Yuxi Xiao, Jason Moffat, Hiromichi Suzuki, Poul Sorensen, Kevin Henry, Chitra Venugopal, Giedre Krenciute, Sheila Singh. Discovery of nuclear envelope mislocalization in MYC-driven group 3 medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5112.

Abstract 5111: Rational synergistic radiosensitization strategies for group 3 medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor. MB is comprised of 4 groups, termed Wnt, Shh, Group 3 and Group 4, each characterized by distinct molecular and clinical profiles. MYC-amplified Group 3 MB is associated imminent fatal metastatic relapse following standard treatment approaches, which currently include surgery, craniospinal irradiation and chemotherapy, highlighting an urgent need for identification of therapeutic vulnerabilities for these tumours. As such, using 4 novel patient-derived models of Group 3 MB that faithfully recapitulate the molecular and histological tumor characteristics, we characterised response to radiation of Group 3 MB in vitro to show that these tumors do not possess intrinsic radiation resistance. Furthermore, cumulative radiation experiments do not increase bulk radiation resistance in MB cells, highlighting the contribution of dynamic cell states to radiation response. In contrast, exposure to radiation leads to changes in cell clonogenic capacity as measured by limited dilution assays. Furthermore, RNA-seq data uncovered altered transcriptomic programs in response to cumulative radiation treatment. In particular, we observe differential regulation of genes encoding potassium ion channels and their regulators as well as genes encoding ECM and cell adhesion proteins. As part of our goal to evaluate the current multimodal treatment strategy of MB, we also show that a set of standard chemotherapeutic agents (etoposide, thiotepa and lomustine) currently used in the clinic do not produce a potent response at optimal doses in vitro, potentially accounting for abysmal survival outcomes seen in chemotherapy treated MB patients. As such, we conducted a screen of 144 FDA approved cancer drugs and identified therapeutics that selectively target Group 3 MB cells in contrast to normal tissue controls (foetal neural stem cells) in the low nanomolar dose range. In particular, topoisomerase inhibitor topotecan produces a potent response specific to MYC-amplified Group 3 MB, and sensitizes 2 patient-derived MB models to gamma irradiation. Altogether, our data characterizes Group 3 MB molecular and cellular responses to current treatment modalities and provides rationale for new clinical strategies to sensitize patients to radiation that will potentially allow to reduce the required radiation doses to treat these aggressive tumors, reduce the incidence of radiation treatment-related morbidity as well as improve cure rates. Citation Format: Julija Povilaikaite, Kaitlin Stitz, Nikhita Austin, Vijay Ramaswamy. Rational synergistic radiosensitization strategies for group 3 medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5111.

Proteomic Profiling of Cerebrospinal Fluid and Its Extracellular Vesicles from Extraventricular Drainage in Pediatric Pilocytic Astrocytoma, towards Precision Oncology.

Pediatric pilocytic astrocytoma (PA) is the most common brain tumor in children. Complete resection provides a favorable prognosis, except for unresectable PA forms. There is an incomplete understanding of the molecular and cellular pathogenesis of PA. Potential biomarkers for PA patients, especially the non-BRAF-mutated ones are needed. Cerebrospinal fluid (CSF) is a valuable source of brain tumor biomarkers. Extracellular vesicles (EVs), circulating in CSF, express valuable disease targets. These can be isolated from CSF from waste extraventricular drainage (EVD). We analyzed the proteome of EVD CSF from PA, congenital hydrocephalus (CH, non-tumor control), or medulloblastoma (MB, unrelated tumoral control) patients. A total of 3072 proteins were identified, 47.1%, 65.6%, and 86.2% of which were expressed in the unprocessed total and in its large-EV (LEV), and small-EV (SEV) fractions. Bioinformatics identified 50 statistically significant proteins in the comparison between PA and HC, and PA and MB patients, in the same fractions. Kinase enrichment analysis predicted five enriched kinases involved in signaling. Among these, only Cyclin-dependent kinase 2 (CDK2) kinase was overexpressed in PA samples. PLS-DA highlighted the inactive carboxypeptidase-like protein X2 (CPXM2) and aquaporin-4 (AQP4) as statistically significant in all the comparisons, with CPXM2 being overexpressed (validated by ELISA and Western blot) and AQP4 downregulated in PA. These proteins were considered the most promising potential biomarkers for discriminating among pilocytic astrocytoma and unrelated tumoral (MB) or non-tumoral conditions in all the fractions examined, and are proposed to be prospectively validated in the plasma for translational medicine applications.

Late relapses in leprosy patients in Brazil: 10-year post-trial of uniform multidrug therapy (U-MDT/CT-BR)

BackgroundLeprosy is a neglected dermato-neurologic, infectious disease caused by Mycobacterium leprae or M. lepromatosis. Leprosy is treatable and curable by multidrug therapy/MDT, consisting of 12 months rifampicin, dapsone and clofazimine for multibacillary/MB patients and for 6 months for paucibacillary/PB patients. The relapse rate is considered a crucial treatment outcome. A randomized Controlled Clinical Trial (U-MDT/CT-BR) conducted from 2007‒2012 compared clinical outcomes in MB patients after 12 months regular MDT/R-MDT and 6 months uniform MDT/U-MDT in two highly endemic Brazilian areas. ObjectivesTo estimate the 10 years relapse rate of MB patients treated with 6 months U-MDT. MethodsThe statistical analyses treated the data as a case-control study, sampled from the cohort generated for the randomized trial. Analyses estimated univariate odds ratio and applied logistic regression for multivariate analysis, controlling the confounding variables. ResultsThe overall relapse rate was 4.08 %: 4.95 % (16 out of 323) in the U-MDT group and 3.10 % (9 out of 290) in the regular/R-MDT group. The difference in relapse proportion between U-MDT and R-MDT groups was 1.85 %, not statistically significant (Odds Ratio = 1.63, 95 % CI 0.71 to 3.74). However, misdiagnosis of relapses, may have introduced bias, underestimating the force of the association represented by the odds ratio. ConclusionsThe relapse estimate of 10 years follow-up study of the first randomized, controlled study on U-MDT/CT-BR was similar to the R-MDT group, supporting strong evidence that 6 months U-MDT for MB patients is an acceptable option to be adopted by leprosy endemic countries worldwide. Trial registrationClinicalTrials.gov: NCT00669643.

Genomic characterization of Mycobacterium lepromatosis from ENL patients from India

BackgroundLeprosy is caused by Mycobacterium leprae and Mycobacterium lepromatosis. Both organisms cannot be cultured in vitro. M. lepromatosis was found to be associated mainly with diffuse lepromatous leprosy and with Lucio's phenomena initially. Later, M. lepromatosis was observed in borderline leprosy cases (BL), lepromatous leprosy cases (LL) and leprosy reactional cases (T1R and ENL). Although many cases are being reported with similar clinical features like Lucio phenomenon in India but M. lepromatosis was not isolated from these cases. The aim of this study was to screen MB patients and patients with type 2 reaction for the presence of M. lepromatosis. MethodologyWe recruited a total of 75 multibacillary leprosy cases (45 MB cases without reaction and 30 type 2 reaction (ENL) cases) from TLM hospitals Purulia (West Bengal), Barabanki (Uttar Pradesh), Shahdara (Delhi) and PGIMER (Chandigarh), India. Punch biopsies of 5 mm were collected in 70% ethanol from all the study subjects. DNA was extracted followed by Hemi-nested PCR targeting 16S rRNA gene specific for M. lepromatosis. Further, PCR products were processed for Sanger sequencing for an absolute confirmation of M. lepromatosis. Whole genome sequencing was done to confirm the presence of M. lepromatosis. ResultWe observed presence of M. lepromatosis in 4 necrotic ENL patients by heminested PCR. There was 100% 16S rRNA sequence similarity with M. lepromatosis FJ924 in one case, 98.96% in two cases and in one case it was 90.9% similarity by nucleotide BLAST (BLASTn) by using the NCBI website. On the basis of Sanger sequencing, we noted presence of M. lepromatosis in 3 necrotic ENL patients as one sample only gave 90.9% similarity by BLASTn. On the basis of de novo assembly and genome obtained, only one sample S4 with a 2.9 mb genome size was qualified for downstream analysis. Sixteen M. lepromatosis- specific proteins were identified in this case and the closest species was M. lepromatosis strain FJ924 based on whole genome level phylogeny. ConclusionThese results provide valuable insights into the prevalence of M. lepromatosis in ENL patients in different regions of India and contribute to our understanding of the genetic characteristics of this pathogen in the context of leprosy.

CSIG-27. FLUID SHEAR STRESS ACTIVATES A MECHANO-METASTATIC CASCADE TO PROMOTE MEDULLOBLASTOMA METASTASIS

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Dissemination of MB cells into cerebrospinal fluid (CSF) and blood initiates metastasis in the central nervous system, which significantly worsens patient prognosis. Disseminated MB cells survive the nutrient-deprived CSF environment and are subjected to fluid shear stress (FSS) by CSF flow. Whether and how FSS contributes to MB metastasis is completely unknown. We computationally simulated FSS dynamics in MB patients using MRI-informed CSF flow models. Based on CSF flow rates and force magnitudes in patients, we engineered two FSS application systems: (1) an orbital shaker system that applies FSS to large populations of cells, and (2) a microfluidic system compatible with live-cell and high-resolution imaging. By applying physiologically relevant FSS to MB cells, we discovered that FSS-treated MB cells metastasize more frequently and form larger metastatic tumors along the spinal cords of mice. Mechanistically, FSS induces actomyosin contraction, which promotes cell clustering, glucose transporter 1 (GLUT1) localization at the cell surface, and GLUT1-dependent glucose uptake. FSS elevates intracellular calcium through mechanosensitive ion channel PIEZO2, which is required for FSS-induced actomyosin contraction, cell clustering, and GLUT1 activity. Genetically perturbing PIEZO2 or pharmacologically inhibiting GLUT1 robustly suppresses MB metastasis in mice. Collectively, we discover that MB cells perceive FSS, define an FSS-activated mechano-metastatic cascade, and demonstrate that this cascade is clinically targetable to mitigate MB metastasis.

MODL-39. ELUCIDATING THE TRANSCRIPTOMIC LANDSCAPE OF METASTATIC PEDIATRIC MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is a highly aggressive and the most common brain tumor in childhood. MB presents a high intertumoral heterogeneity, with at least four molecular subgroups (SHH, WNT, Group 3, and Group 4) identified. MB can metastasize to the leptomeningeal space, known as Leptomeningeal Disease (LMD) and its presence is a universal predictor of poor outcome among MB patients. Metastatic MB is predominantly found in the MB Group 3 type. Although LMD represents a main clinical challenge, its molecular mechanisms remain poorly characterized. Recent research has shown that primary and MB metastasis diverge dramatically. Our work has focused on establishing therapy naïve Group 3 Patient-Derived Xenografts that faithfully replicate the nature of primary and metastatic Medulloblastoma with the aim to perform comparative genomic and transcriptomic analyses between these models to identify genetic drivers/pathways that sustain metastatic MB or leptomeningeal disease. Our results show various signaling pathways enriched across LMD models, such as protein secretion and oxidative phosphorylation. We also have identified differentially expressed genes, where sets of genes have shown to be present in more than one PDX model, such as members of the Solute Carriers family (SLC44A3 and SLC17A9) and FCGBP. Retrieval of short variants from RNAseq data has not revealed thus far mutations enriched in LMD, suggesting these changes could be attributed to epigenetic disruption rather than genetic changes. In conclusion, our results support the notion that primary and LMD are transcriptionally different, with various enriched pathways and sets of DEG among LMD Group 3 PDX models. These findings are in progress for functional validation. Work is currently being done to profile the epigenome of LMD by correlating transcriptomic data with active chromatin markers such as H3K27ac and H3K4me1. Through these approaches, we aim to elucidate the genetic dependencies of metastatic Medulloblastoma that will help for targeted therapies.

Feasibility, safety and prognostic relevance of acetylcholine provocative testing in patients with myocardial bridge: a prespecified analysis of the RIALTO Registry

Abstract Introduction Myocardial bridge (MB) is the most frequent inborn coronary artery variant. Although MB has long been considered a benign entity, a growing body of evidence has suggested its association with angina and adverse cardiac events. However, to date, no data on long-term prognosis are available, nor on therapies improving cardiovascular outcomes. We are currently conducting an ambispective, observational, multicentre, study in which we enroll patients with a clinical indication to undergo coronary angiography (CA) and evidence of MB, aiming to describe the incidence of symptoms and cardiovascular events at baseline and at long-term follow-up (FUP). The role of invasive full-physiology assessment in modifying the discharge therapy and eventually the perceived quality of life and the incidence of major cardiovascular events will be analysed. Herein, we assessed the safety, feasibility, and the prognostic role of invasiveacetylcholine (Ach) testing in unmasking coronary artery spasm in patients with MB. Materials and Methods A total of 444 patients with MB were consecutively enrolled in the RIALTO registry. 73 underwent intracoronary Ach testing. Both patients with acute and chronic presentations were included. The decision to perform the provocative testing was left to the operator's judgment and performed according to a standardized protocol. The incidence of major and minor complications during Ach testing was recorded. Furthermore, the incidence of major adverse cardiac events (MACE), defined as the composite of cardiac death, myocardial infarction, and cardiac hospitalization, and the rate of patients with a Seattle Angina Questionnaire (SAQ) Angina Summary Score <70 were compared between patients with and withoutcoronary artery spasm. Results Epicardial spasm was diagnosed in 58.9% of MB patients, whereas microvascular spasm in 6.9% according to COVADIS criteria. The most frequent location of coronary artery spasm (CAS) was the intramural segment. Regarding safety, no fatal or major non-fatal events occurred. The rate of minor side effects was low (12.3%) and comparable between acute and chronic patients. MACE occurred in 12 (18.8%) patients, without significant differences in the rate of MACE between the study groups (18.2% in patients with a positive Ach testing vs. 20% in patients with a negative Ach testing; log-rank = 0.88). However, the rate of significant angina (SAQ < 70) was higher in the Ach+ group compared with the Ach- group (47.7% vs. 25%; log-rank = 0.04). Conclusions CAS often coexists with MB. The execution of Ach testing in this cohort proved to be safe and feasible, identifying a subgroup of patients with a higher angina burden and poor quality of life, although with a comparable rate of MACE at 2 years. In our study, invasive provocative test improved the ability to unmask vasomotor disorders associated with MB and to prescribe a tailored therapy with calcium antagonists, which improved symptoms.

JS07.5.A PROGNOSTIC FACTORS ASSOCIATED WITH POOR OUTCOME IN ADULT PATIENTS WITH MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma (MB) is a rare tumor in adults. Current recommendations are based on scarce data available in adults, and mostly derived from pediatric series. Prognostic factors have not been uniformly defined in adults to date. MATERIAL AND METHODS We retrospectively collected all cases of adult patients diagnosed and treated between 1995 and 2019 in our institution for histopathological proven MB. We described patient´s characteristics, disease features, treatment modalities and studied the prognostic factors for survival outcomes using univariate and multivariate analysis. RESULTS We identified a total of 89 cases. Male gender (66%), lateral cerebellar location (48%), desmoplastic/nodular histology (43%), Sonic Hedgehog molecular group (76%) and Chang´s M0 (72%) were the most frequent patient´s and disease characteristics. Patients mostly achieved complete resection/near total resection (62%); surgery followed by chemotherapy/radiotherapy (CT/RT) was the most frequent initial strategy (76%), favoring carboplatin-based regimens (70%), posterior fossa RT ≥50 Gy (71%) and craniospinal RT ≥ 30 Gy (59%) as the most frequently regimen used. According to the Association of French Speaking Neuro-oncologists and the European Reference Network on Rare Adult Cancers risk scales (ANOCEF and EURACAN), we found 44 and 48% of intermediate-risk patients and 49 and 45% of high-risk patients, respectively. Both cerebral and spinal presentation at diagnosis was documented in 16 (18%) patients. Complete response was achieved in 71(80%) patients. Nine was the maximal number of progressions documented, 35(39%) patients relapsed, CT was the preferred strategy at relapses, privileging carboplatin-based regimens at first relapse. The median overall survival (mOS) was 124.42 months (95%CI 68.53 - 180.11) and the median progression free survival (mPFS) was 30.48 months (95%CI 13.46 - 47.51), the 5-year OS was 53% and the 5-year PFS was 51%. In multivariate analysis, Chang´s ≥ M2 metastatic classification (p=0.001), no CT in association to RT (p=0.005), and lower doses on craniospinal RT <30 Gy (p=0.015) were associated with poor prognosis for OS; Chang´s ≥ M2 (p=0.009) was also a prognostic factor for poor PFS. CONCLUSION This study dedicated specifically to adult MB patients suggests relevant prognostic factors for adult MB patients. We identify Chang´s ≥ M2 as poor prognostic factor of OS and PFS, and we confirm that the use of CT/RT at diagnosis and RT doses ≥ 30 Gy are associated with better survival in adults MB.

P04.08.A PATIENT-DERIVED ORGANOIDS FOR MODELING AND CHARACTERIZING PEDIATRIC BRAIN TUMORS

Abstract BACKGROUND Central nervous system (CNS) tumors are the most common solid tumors in the pediatric population and the leading cause of death among childhood cancers. They present common features related to the development and produce symptoms based on their location, rate of tumor growth and age of the child. Tumors of embryonal origin, such as medulloblastoma or primitive neuroectodermal tumor, have a higher incidence in younger patients, whereas older ones tend to present tumors of glial origin. Most of CNS-embryonal tumors are highly malignant and consequently leading to poor prognoses. Despite the understanding of their underlying molecular landscape that has helped in clinical advances to improve life expectancies of the patients, there is still an emerging need to minimize morbidity and manage the long-term therapy effects. MATERIAL AND METHODS Human samples were collected according to protocols approved by the institutional review board with written consent. Patient-Derived Organoids (PDOs) and Patient-Derived Xenograft Organoids (PDXOs) were obtained using scalpels by chopping in small pieces the tumor tissue and were grown in PDOs medium. PDOs and PDXOs were analyzed through DNA methylation, immunostainings, sc-RNA sequencing and orthotopic nude mice engraftment. Therapeutic regimens were adapted according to patients’ treatment protocols depending on the tumor of origin. RESULTS Developing new models to better understand the complexity and heterogeneity of pediatric tumors and to test personalized treatment strategies remains a clinical challenge. We tried to answer this medical need with PDOs and PDXOs established through the direct in vitro culture of different kinds of primary tumors (ependymoma EPN, medulloblastoma MB and low-grade glial tumors) and MB patient derived xenograft (PDX)-derived tumors. We have developed a method that recapitulates mutational profiles, histological features, and cellular heterogeneity of the corresponding parental tumors. We have obtained promising results from the DNA methylation profile analysis, nowadays used for an accurate routine CNS tumors diagnosis. Through this new model, we confirmed the action of clinically used therapeutic regimens, i.e., for EPN with ZFTA-RELA fusion and G3 MB-derived PDOs. PDOs were subjected to the same treatment the patients underwent and responded similarly to the patients. CONCLUSION We found a suitable in vitro culture condition to generate and maintain PDOs from different kinds of pediatric brain tumors. Their reliability has been proved by different analyses, confirming they still resemble the parental tumors. Through this new model, we confirmed the action of clinically used therapeutic regimens, suggesting its potential at the translation level as a reliable tool for testing personalized medicine, drug screening and development of newer and more specific therapeutic strategies.

P04.07.B CHARACTERIZATION OF ARHGAP36/MYC-INDUCED GROUP 3 MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma (MB) is one of the most common pediatric brain cancers of the central nervous system. It is stratified into 4 molecular subtypes (WNT, SHH, group 3, and group 4), which differ from one to the other for various features, such as cellular morphology, age of onset, set of mutations, and percentage of survival. A common point between the four MB subgroups is the absence of treatments for this disease. The current gold standard treatment has deleterious side effects for the patients, which spans from growth defects to low hormone levels and problems with learning. Analyzing transcriptomic data of group 3 MB patients we found ARHGAP36 as a possible gene involved in tumor formation or propagation. Rho GTPase-Activating Protein 36 (ARHGAP36) is a gene that plays a role in regulating PKA activity. Specifically, the ARHGAP36 protein can inhibit PKA function through two distinct mechanisms. Firstly, it acts as a pseudosubstrate for the catalytic subunit of the PKA protein. Secondly, it can drive protein degradation via a lysosome-mediated process. MATERIAL AND METHODS We took advantage of the Piggybac/transposon technology to induce a strong and stable overexpression of our genes of interest to generate two different models, one in-vivo and one in-vitro with induced pluripotent stem cells-derived human cerebellar organoids. We also exploited the cre/LoxP system in transgenic mice to overexpress ARHGAP36 under the control of specific promoters. The analysis was conducted using standard techniques such as RT-qPCR and Western Blot, together with immunofluorescence of the cerebellar section and live imaging of the modified cerebellar organoids. The gene expression profile of the tumors obtained in mice was analyzed by RNA sequencing and compared to known group 3 MB mouse models. RESULTS We analyzed RNA-sequencing data from medulloblastoma patients and found that high expression of ARHGAP36 correlated with poor prognosis in patients. Through PKA inhibition, ARHAP36 induces ectopic activation of SHH signaling and cell proliferation leading to abnormal development of the cerebellum in mouse embryos. Notably, despite its inductive activity on the SHH pathway, ARHGAP36 expression is higher in group 3 and group 4 MB, rather than in the SHH subgroup. In this study, we demonstrate that intracranial transfection of P0 mouse pups with plasmids overexpressing MYC and ARHGAP36 can generate group 3 medulloblastoma in-vivo. We studied and then confirmed the involvement of the binding with PKA as a tumorigenic mechanism in the MYC/ARHGAP36 co-overexpression using several mutated forms of the gene. We studied the obtained tumors for the presence of group 3 MB immunophenotypical markers, as well as their gene expression profile by RNA-seq. CONCLUSION These findings can become of key interest as possible druggable targets to efficiently treat group 3 medulloblastoma patients in the future.

Analgesic efficacy of bupivacaine with or without magnesium adjunct in bilateral ilioinguinal and iliohypogastric nerve blocks following cesarean section under subarachnoid block: A randomized controlled trial.

Pain relief is a basic aspect of anesthesia care. Optimal post-cesarean section analgesia should minimize postoperative complications, facilitate infant care, and prevent postoperative morbidity. Bilateral ilioinguinal-iliohypogastric (IIIH) nerve block has been considered as a part of the multimodal approach. This study was designed to explore the efficacy of adding magnesium sulfate as an adjunct to bupivacaine to provide analgesia following cesarean delivery, using bilateral IIIH nerve block. Seventy-two parturients who were scheduled for elective cesarean section were randomized into two groups of 36 patients each. Group MB patients were given bilateral IIIH nerve block with 250 mg of magnesium sulfate and 95 mg of 0.25% plain bupivacaine. Group B patients were given bilateral IIIH nerve block with 0.9% normal saline and 95 mg of 0.25% plain bupivacaine. Postoperative visual analog scale (VAS) pain scores at post-anesthesia care unit (PACU), 2, 4, 6, 12, and 24 h, both at rest and with activity were measured. Rescue doses of opioid (intravenous [IV] tramadol 50 mg) at each time point of assessment, total tramadol consumption, time to patients' first request for rescue analgesic agent, and patients' satisfaction score were recorded. The demographic and social parameters of patients in both the groups were comparable. The mean postoperative VAS pain score in group MB was significantly lesser both at rest and with movement than in group B at 12 and 24 h after surgery (P < 0.05). Total postoperative tramadol consumed over 24 h was significantly lesser in the magnesium group than in the control group (125.55 ± 20.76 vs. 160.24 ± 25.82 mg), with a P value of 0.026. The time to patient's first analgesic request was significantly prolonged in group MB compared to group B (505.2 ± 41.4 vs. 372.6 ± 88.8 min, respectively), with a P value of 0.040. However, patients in both groups expressed good satisfaction scores. Magnesium sulfate as an adjunct to bupivacaine in bilateral IIIH nerve block reduced VAS pain scores, total tramadol consumption, and prolonged post-cesarean section analgesia.

MDB-27. A PRO-DRUG OF TRIPTOLIDE IN CLINICAL DEVELOPMENT ATTENUATES HIGH-RISK MEDULLOBLASTOMA GROWTH

Abstract Medulloblastoma (MB), the most common malignant brain tumor of childhood, is currently classified in four major subgroups. A natural compound used in Chinese medicine for centuries, triptolide, has been shown to have a potent antitumor activity, and the efficacy of one of its derivatives is being evaluated in the clinic. However, to date no preclinical data supports the use of triptolide for the treatment of patients with MB. Here, we performed bioinformatic analyses to predict which subgroup of medulloblastoma patients would more likely benefit from the use of triptolide, and validated its efficacy in series of ex vivo and in vivo mouse and human derived MB models. Correlation analyses identified Group 3 (G3) MB patients as the most prone to respond to triptolide treatment. Consistent with this prediction, triptolide attenuated the growth of G3 MB at lower doses than those classified as SHH subgroup, increasing symptom-free survival in G3 mouse MB models. Transcriptomic analyses revealed MYC signaling as the key downstream target of triptolide in MB cells. Importantly, a pro-drug of triptolide in clinical development, minnelide, similarly showed efficacy in mouse and human derived G3 in vivo MB models. Our findings highlight the potential of repurposing Minnelide to treat G3 MB patients. Due to their dismal prognosis, inclusion of G3 MB patients in future minnelide clinical trials should be considered.

MDB-39. ASSESSMENT OF MEDULLOBLASTOMA PATIENTS THROUGH MONITORING EXTRACELLULAR VESICLES VIA SURFACE-ENHANCED RAMAN SPECTROSCOPY COMBINED WITH MACHINE LEARNING

Abstract Medulloblastoma (MB) is the most common brain pediatric tumor associated with considerable morbidity. While MB is treated with multimodal approaches, including surgery, the currently available technologies fail to provide non-invasive access for continuous assessment of disease progression and therapy efficacy. There is a need to develop monitoring systems for frequent use with minimum invasiveness that provides actionable information incorporating the heterogeneous nature of MB. Liquid biopsy is a non-invasive approach using circulating biomarkers, including nano-sized extracellular vesicles (EVs). EVs are secreted by all cells, even cancerous ones. Molecular composition of cancer EVs contains fingerprints of their parental cell, reflective of salient features of the underlying disease. We used 54 pediatric plasma samples of MB patients to determine feasibility of assessing disease progression through EVs analysis. EVs were isolated from plasma using size exclusion chromatography prior to loading onto unique MoSERS chip. MoSERS platform contains nanostructured capture element for single EV detection capable of profiling liquid biopsy samples for surface-enhanced Raman Spectroscopy. A total of 54 datasets were generated, each comprised of EV spectra collected with the MoSERS chip at a single EV resolution. A spectral library was integrated with the datasets of patients with confirmed MB diagnosis (n=34) and healthy controls (n=20), required to train and test a machine-learning implementation. The combination of MoSERS with robust machine learning algorithm provides a strong analysis and a simple interpretation of the patient’s health status. We optimized a pipeline for the data collection and analysis, demonstrating the feasibility of the MoSERS platform for pediatric cancer indication and need for less than 10 μl of sample to generate each unique dataset. Preliminary results demonstrate that MoSERS performance correlates with the results of clinical standards, providing a proof-of-concept for its implementation as a non-invasive and accessible alternative to monitor the health status of MB patients.

MDB-43. GRADIENT ENTROPY-BASED RADIOMIC RISK-SCORE FROM T1-WEIGHTED PRE-TREATMENT MRI SCANS CAN PREDICT SURVIVAL IN PEDIATRIC MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma (MB) is the most frequent malignant brain tumor in children with an inadequate 5-year survival rate of 70-75%. A key determinate in MB treatment (consisting of radiation, chemotherapy) is the accurate risk-stratification of patients into low- and high-risk. The current stratification approaches (Chang’s classification, molecular categorization) are sub-optimal in predicting individual patient outcomes. Reliable risk-assessment approaches can significantly impact treatment management. Our group previously developed a new radiomics feature COLLAGE that measures the orientation of local intensity gradients as an estimate of lesion heterogeneity. We hypothesize that the disorder (i.e., heterogeneity) in orientation of local per-voxel intensity measured by COLLAGE, will tease-out differences in patients with low - versus high-risk MB tumors. METHODS T1-weighted MRI scans of 49 pediatric MB patients were retrospectively collected from Children’s Hospital Los Angeles and Cincinnati Children’s Hospital Medical Center. Registration of the scans was performed via age-specific pediatric atlases, followed by skull stripping and bias correction. Ground truth annotations for tumor sub-compartments; enhancing tumor, edema, and non-enhancing tumor, were generated by two experienced radiologists. Then, 52 Collage features were extracted from each of these sub-compartments, followed by conducting a Lasso model and a cox-proportional hazards model to identify the top features for risk-stratification. A continuous risk score was created for every patient and a risk threshold was obtained from the fitted cox model to stratify patients into low- and high-risk. RESULTS Our risk-score consisted of 13 top COLLAGE features from edema and 14 from non-enhancing tumor sub-compartments, respectively, and demonstrated significant differences between low- and high-risk patients (p = .017 for edema, p =0.0027 for non-enhancing tumor). CONCLUSION Our preliminary analysis suggests that gradient-entropy radiomic risk score maybe able to predict overall survival in pediatric MB tumors. In future, we plan to include additional patients to perform independent multi-site analysis.

MDB-21. THE OTX2-REGULATED ALTERNATIVE SPLICING LANDSCAPE CONTROLS DIFFERENTIATION AND RECONSTITUTES THE DEVELOPMENTAL ORIGINS OF GROUP 3 MEDULLOBLASTOMA

Abstract 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-16. TARGETING EPIGENETIC DYSREGULATION IN PAEDIATRIC BRAIN TUMOURS

Abstract Medulloblastoma (MB) is the most common paediatric malignant brain tumour and is classified into four distinct molecular subgroups (WNT, SHH, G3 and G4), each of them further divided into subtypes with different prognosis and responses to therapy. Deregulation of chromatin modifier genes play an essential role in MB, particularly in the G4 subgroup. A BMI1High;CHD7Low molecular signature identifies patients with poor survival. We have shown that BMI1High;CHD7Low sustains MB growth through regulation of MAPK/ERK signalling and via epigenetic regulation of inositol metabolism in both G4 MB cells and patients. These tumours display over-activation of MAPK/ERK and AKT/mTOR pathways leading to energetic rewiring characterised by enhanced glycolytic capacity and reduced mitochondrial function. We demonstrate that inositol administration counteracts this metabolic alteration and significantly extends survival in an in vivo pre-clinical model. Additionally, we identify a synergistic vulnerability of G4 MB to a combination treatment with BMI1 and MAPK/ERK inhibitors that overcomes the acquired resistance induced by single drug therapies. Importantly, we have now analysed recurrent G4 MB and found that the BMI1High;CHD7Low signature is maintained at recurrence and it continues to predict the pharmacological vulnerabilities we previously described. Finally, we extended the analysis to other paediatric brain tumours, including histone mutant gliomas and ependymomas, and identified molecularly defined subgroups that exhibit differential response to the compounds predicted to be effective by the BMI1High;CHD7Low signature, hence expanding the spectrum of tumour potentially amenable to these novel pharmacological approaches.