Embryonal Tumor With Multilayered Rosettes Research Articles

CSIG-33. CERAMIDE-1-PHOSPHATE/CERK ARE REGULATORS OF ETMR GROWTH AND POTENTIAL THERAPEUTIC TARGETS

Abstract Embryonal tumor with multilayered rosettes (ETMR) is a highly aggressive CNS neoplasm which occurs almost exclusively in infants and is associated with an extremely poor prognosis. To identify functional signaling lipids that contribute to the aggressive nature of ETMR and that can be targeted therapeutically, we carried out mass spectrometry imaging on human ETMR patient samples, the patient-derived cell line BT183, and normal neural stem cells. We identified an accumulation of ceramide-1-photphate (C1P) within the rapidly proliferating embryonal tumor cells in patient samples and the cell model. We also detected high accumulation of C1P within the aberrant vascular proliferations in patient samples. Ceramide-1-phosphate is a pleiotropic bioactive signaling lipid that controls cell fate. C1P and its synthesizing enzyme, ceramide kinase (CERK), are known mitogens that have been shown to regulate cell proliferation in several non-CNS cancers. C1P is known to mediate the upregulation of PI3K/Akt/mTOR, MAPK/MEK/ERK1/2, Rho kinase, JNK, to promote cell survival and migration. After spatially mapping C1P accumulation in patient samples and our in vitro 3D models, we next carried out IHC to correlate lipid accumulation with its synthesizing enzyme, CERK. CERK was found to be abundantly accumulated in both endothelial and tumor cells within the tumor microenvironment of patient samples. Western blot analysis of CERK demonstrated a significant increase in the ETMR 3D tumorspheres compared to the NSC neurospheres. Treatment of the ETMR 3D tumorspheres with the CERK inhibitor, NVP-231, potently reduced cell growth and viability. Western blot analysis of ETMR tumorspheres at 8 and 24 hours post-treatment demonstrated a time-dependent decrease in N-MYC and a subsequent increase in p53. Studies are ongoing to validate these findings in vivo and to elucidate the signaling pathways that C1P/CERK regulate in both the vascular and tumor cells in ETMR.

INNV-24. CONGENITAL EMBRYONAL TUMOR WITH MULTILAYERED ROSETTES AND TREATMENT IN A NEWBORN

Abstract BACKGROUND Embryonal Tumor with Multilayered Rosettes (ETMR) is a rare but deadly pediatric brain tumor with no standard of care treatment. We present a unique case of congenital ETMR and treatment strategy using intrathecal topotecan and no radiation therapy. Case report: A term baby was admitted to NICU at 12 hours of life with respiratory arrest requiring intubation. CT of the brain revealed a 4.2x3.9x3.5cm left posterior fossa hyperdense hemorrhagic mass with moderate hydrocephalus. She underwent posterior fossa craniotomy for biopsy and evacuation at two days of life. Pathology was consistent with an ETMR. MRI brain after first resection was not concerning. She underwent re-resection at 3 months of age due to re-growth (2x3x3cm). She was then treated as per Head Start 4 chemotherapy regimen with vincristine, cisplatin, etoposide, cyclophosphamide, and methotrexate, followed by consolidation with three tandem auto-hemopoietic stem cell transplants (HSCT) with carboplatin and thiotepa conditioning. Maintenance treatment consisted of intrathecal topotecan every four weeks (0.2mg per dose <1 year old, 0.25mg per dose when ≥1 year old) for 12 doses. She also received oral vorinostat (100mg PO QD) and isotretinoin (80mg/m2 divided BID) during maintenance for 1 year. No radiation was given. The treatment course was complicated by mucositis, febrile neutropenia, pseudomonas sepsis and an episode of respiratory arrest secondary to choking from epistaxis. The patient is currently doing well and continues to remain in remission for over seven months after end of treatment. CONCLUSION We present a case of congenital ETMR with a unique treatment option and strategy using Head Start 4 therapy with three tandem auto-HSCT as consolidation, followed by 12 months of maintenance therapy with intrathecal topotecan, oral vorinostat, and isotretinoin, without any radiation, leading to sustained remission.

Decoding pediatric spinal tumors: a single-center retrospective case series on etiology, presentation, therapeutic strategies, and outcomes

IntroductionSpinal tumors (ST) often result in dire prognosis, carrying risks such as permanent paralysis, sensory loss, and sphincter dysfunction. Data on their incidence and etiology in pediatric populations are markedly scant. Our study investigates the etiology, clinical manifestation, treatment, and outcomes of pediatric ST.MethodsWe conducted a retrospective review of our institutional pediatric oncology and neurosurgery database, examining 14 patients under 18 years admitted with ST due to oncological diseases since 2005. We analyzed the clinical presentations, evaluations, molecular diagnostics and treatments for these patients.ResultsThe study spanned 15 years and included 14 pediatric patients, each diagnosed with distinct spinal tumor entity. The mean patient age was approximately 19.6 ± 10.1 months. Severe axial pain along the vertebral column was observed in 13 patients, while acute neurological deterioration manifested in 7 patients. As a first-line intervention, 13 patients underwent decompressive surgery through laminectomy and tumor resection, and only one patient received chemotherapy solely. Before surgery, seven patients were unable to walk; post-surgery, six of them regained their ability to ambulate. The diagnosis encompassed a range of neoplasms: two instances of Ewing sarcoma, 3 instances of teratoma, one case presenting an atypical teratoid Rhabdoid tumor, two instances each of low-grade astrocytoma and neuroblastoma, and single instances of ependymoma, meningioma, rhabdomyosarcoma, and embryonal tumors with multilayered rosettes (ETMRs). Three patients succumbed two years after initiating therapy.ConclusionDespite their rarity, intraspinal tumors in pediatric patients pose substantial therapeutic challenges. The intertwined complexities of the disease entity and the patient’s neurological status demand swift initiation of an individualized therapeutic strategy. This crucial step helps optimize outcomes for this patient cohort, who frequently grapple with debilitating health conditions. Inclusion of these patients within a registry is mandatory to optimize treatment outcomes due to their rarity in pediatric population.

Cortically-based Brain Tumors in Children: A Decision-tree Approach in the Radiology Reading Room.

Cortically-based brain tumors in children constitute a unique set of tumors with variably aggressive biological behavior. As radiologists play an integral role on the multidisciplinary medical team, a clinically useful and easy-to-follow flowchart for the differential diagnoses of these complex brain tumors is essential.This proposed algorithm tree provides the latest insights into the typical imaging characteristics and epidemiologic data that differentiate the tumor entities, taking into perspective the 2021 World Health Organization's classification and highlighting classic as well as newly identified pathologic subtypes using current molecular understanding.ABBREVIATIONS: Astroblastoma=AB) Angiocentric glioma (AG) Atypical teratoid rhabdoid tumor (ATRT) Central Nervous System tumor (CNS) CNS neuroblastoma FOXR2-activated (NB-FOXR2) Desmoplastic infantile glioma/astrocytoma (DIG/DIA) Diffuse hemispheric glioma, H3 G34-mutant (DHG) Diffuse glioneuronal tumor with oligodendroglioma-like features and nuclear clusters (DGONC) Dysembryoplastic neuroepithelial tumor (DNET) Embryonal Tumors with Multilayered Rosettes (ETMR) Ependymoma (EP) Focal cortical dysplasia (FCD) Ganglioglioma/gangliocytoma (GG) Infant-type hemispheric glioma (IHG) Intracranial pressure (ICP) Long-term epilepsy-associated tumors (LEATs) Pediatric diffuse low-grade gliomas (pLGG) MR spectroscopy (MRS) Multinodular and vacuolating neuronal tumor (MVNT) Overall survival (OS) Pediatric diffuse high-grade gliomas (pHGG).

ETMR-06. PNOC031 - INTERNATIONAL TRIAL FOR EMBRYONAL TUMOR WITH MULTILAYERED ROSETTES

Abstract Embryonal tumors with multilayered rosettes (ETMR) are aggressive central nervous system neoplasms with unfavorable prognosis, exhibiting rapid disease progression and frequent post-therapeutic relapse. The rarity of ETMR necessitates global collaboration to advance therapies. Radiotherapy (RT) has been associated with improved survival outcomes in ETMR with case series revealing that nearly half of ETMR patients treated without RT relapse within six months of diagnosis. However, RT is often withheld due to young patient age and neurodevelopmental considerations. This high-rate of on-treatment relapse is unique to ETMR and suggests that radiation-sparing regimens may be suboptimal. PNOC031 is an international collaboration, establishing a global clinical trial to innovate and improve therapeutic interventions for children with ETMR. Eligible patients with newly-diagnosed ETMR are stratified into three cohorts: Cohort 1 - gross total resection/RT-eligible patients to receive early focal radiotherapy (RT); Cohort 2 – gross total resection/not RT-eligible to receive high-dose chemotherapy with autologous stem cell rescue; and Cohort 3 – less than gross total resection or with metastatic disease to receive RT or high-dose chemotherapy per RT eligibility, based on age and tumor location. Cohort 1 will test the hypothesis if early RT improves outcomes in a pilot cohort of 20 patients. Patients will receive six weeks of induction chemotherapy, followed by focal irradiation and an additional six weeks of chemotherapy. Six-month progression-free survival will be assessed to determine the impact of radiotherapy on the rate of early relapse. Cohorts 2 and 3 will allow collection of uniform prospective clinical data using a common treatment backbone, to inform future clinical trial design for ETMR. Incorporating a myriad of correlative studies, including next-generation sequencing, cerebrospinal fluid analyses, microbiome profiles, and evaluations of social determinants of health alongside cognitive outcomes, will enhance comprehensive understanding of clinical responses and advance therapeutic options for this high risk disease.

ETMR-05. DEVELOPMENT OF NOVEL PRECLINICAL MODELS AND THERAPEUTIC STRATEGIES FOR ETMR

Abstract BACKGROUND Embryonal Tumor with Multilayered Rosettes (ETMR) is a very rare, aggressive pediatric brain tumor with a dismal 5-year overall survival rate. Few genetic aberrations in miRNA clusters and processing machinery have been detected in ETMRs with 90% of ETMR patients having the C19MC amplification. ETMRs show unique histopathological features which, together with the detection of C19MC or DICER alterations, are necessary for diagnosis. The cellular and molecular mechanisms underlying ETMR development are poorly understood, and targeted therapeutics have not been developed due to scarcity of preclinical models. To expand knowledge of ETMR biology and accelerate the design of novel and therapies, it is thus essential to develop preclinical models. METHODS We have generated two novel patient-derived ETMR cell lines from resected patient derived tumor samples (CTBP-03A and CBTP-139) and created three patient-derived xenograft (PDX) models (CTBP-03A, CBTP-139 and CBTP-38). We have used these unique patient-derived cell lines to identify mechanisms driving ETMR development and progression. To identify novel therapeutic targets, we conducted high-throughput drug screening (HTDS) utilizing 2480 approved and investigational drugs, against the CBTP-03A and CBTP-139 lines. RESULTS Among the candidate drugs showing an inhibitory effect on both lines, compounds were selected based on their activity, CNS penetration ability, mechanism of action, and bioavailability, if known. The top selected hits were screened in an all-versus-all format, totaling 1953 combinations. The rapidity of action and efficacy of the top choice compounds used as single agent or in double and triple combination was assessed by caspase glow and cell titer glow assays. This initial combination screen identified DNA replication stress, retinoid receptor agonist and FGF signaling as potential combinatorial targets. Data are currently being validated in vivo. CONCLUSIONS This is the first combinatorial HTDS on two novel patient-derived cell lines which highlighted potential drug combination strategies for the treatment of ETMR.

ETMR-08. DECODING TUMOR-NEURONAL COMMUNICATION IN PEDIATRIC BRAIN TUMORS: INSIGHTS FROM ETMR 3D MODEL REVEALING MECHANO-SIGNALING DYNAMICS AND THERAPEUTIC OPPORTUNITIES

Abstract INTRODUCTION Exploring tumor-neuronal interactions is essential for comprehending brain tumor proliferation, chemotherapy resistance, and patient survival. While glioma research has made significant strides, our understanding of tumor-neuronal interactions in pediatric brain tumors, notably Embryonal Tumors with Multilayered Rosettes (ETMRs), remains limited. METHODS To elucidate tumor-neuronal interactions in ETMRs, we developed a 3D model by co-aggregating the human ETMR cell line BT-183 with human induced pluripotent stem cells (iPSCs) and allowing them to mature into forebrain organoids for 30 days, forming ETMR-forebrain organoids (ETMR-FBO). We further employed single-cell RNA sequencing to compare the gene expression profile of healthy forebrain organoids (FBO) with the ETMR-FBOs. Additionally, we conducted immunohistochemistry of 3D model sections for comparative evaluation and spatial transcriptomics of murine and human primary ETMR samples. RESULTS Our integrated dataset of 48,764 cells of healthy FBOs (n = 4) and ETMR-FBOs (n = 6) revealed that ETMR tumor cells impeded the neuronal differentiation of the FBO cells. The ETMR-FBOs exhibited a significant enrichment of neuro-stem cells (67% vs 49%), accompanied by a reduction of more differentiated neuronal cells. Comparative trajectory analysis confirmed a blockage of neuronal maturation/differentiation in ETMR-FBOs compared to controls without ETMR cells. Immunohistochemistry further confirmed an enrichment of SOX2-positive/synaptophysin-negative neuronal progenitors in the organoid areas closest to the tumor border. Additionally, we identified upregulated extracellular matrix-related pathways, particularly integrin and YAP/TEAD signaling, in the neuro-stem cell compartment of ETMR-FBOs. This implies a tumor-specific mechano-signaling mechanism influencing the neuronal differentiation state of the tumor microenvironment. CONCLUSION Our findings suggest that mechano-signaling-related tumor-neuronal interactions in ETMR contribute to the formation of an immature neuronal niche. Targeting mechano-signaling may hold promise for innovative therapies by disrupting tumor architecture and enhancing susceptibility to chemotherapy.

ETMR-18. ADVANCING ETMR RESEARCH: NOVELIN VITRO ANDIN VIVO PRECLINICAL MODELS FOR DEVELOPING MORE EFFECTIVE TREATMENTS

Abstract BACKGROUND Embryonal Tumor with Multilayered Rosettes (ETMR) is a rare and aggressive tumor that occurs mainly in infants under three years of age. Genetically, ETMRs are characterized by amplification and fusion of a microRNA cluster on chromosome 19 (C19MC) with TTYH1, present in ~90% of the tumors. ETMR patients still have a poor prognosis without any recent advances in treatment. A major limitation to develop more effective treatment strategies is the lack of sufficient preclinical models. METHODS Establishment of ETMR patient-derived xenograft (PDX) models and tumoroid lines, molecular characterization, high throughput in vitro drug screens, targeted drug treatments in vitro and in vivo. RESULTS We have established four new tumoroid and five new PDX ETMR models. These models faithfully recapitulate ETMR patients’ tumors based on their histological, transcriptomic, and methylome signatures. The in vitro models were derived from two patients and two PDX models and included three C19MC-amplified cell lines, of which two were a primary-relapse pair, and one a primary C19MC non-amplified model. In vivo we established the same models as PDX models subcutaneously plus an additional C19MC-amplified relapse model. High-throughput drug screening assays, using drug libraries with 225 clinically approved drugs and other small molecules, were conducted across the panel of in vitro models. Potential therapeutic candidates with promising activity in ETMR tumoroid lines identified from these screens include FGFR, XPO1, MTOR, MDM2 and topoisomerase inhibitors. CONCLUSIONS The establishment of novel preclinical models represents a significant advancement in ETMR research, offering a platform that captures the molecular complexity of the disease. The identification of several inhibitors shows the potential of these models for drug discovery and preclinical testing. Ongoing in vitro and in vivo experiments with these drugs and combinations aim to further validate their activity, moving towards the development of more effective therapies for ETMR patients.

MODL-01. IN VITRO MODELING OF AND AUTOMATED DRUG SCREENING IN EMBRYONAL BRAIN TUMORS USING TUMOR-BRAIN ORGANOIDS

Abstract BACKGROUND Embryonal brain tumors (EBTs) such as medulloblastoma, atypical teratoid rhabdoid tumor (AT/RT), and embryonal tumor with multilayered rosettes (ETMR) uniquely arise in the developing brain. Traditional 2D models often fall short in capturing the tumoral microenvironment (TME) and intratumoral heterogeneity, which may limit their translational potential. METHODS To address this, we co-aggregated EBT cells from AT/RT (Chla02 and SHH310) and ETMR (BT183 and murine GEMM cells) with forebrain organoids derived from human induced pluripotent stem cells and developed an automated, screening-ready in vitro model (EBT-FBO). We used immunohistochemistry, whole-mount immunostaining, and single-cell RNA sequencing to characterize our model and compared it with a developmental forebrain atlas. We further used our model for cell type-specific drug screening and validated the results in vivo. RESULTS Our EBT-FBO model mimicked the differentiation states of a mid-gestational fetal forebrain, providing a natural immature neuronal environment for supratentorial EBT cells. This environment allowed the EBT cells from both AT/RT and ETMR to mimic in vivo tumor cell states and avoided the cycling cell selection common in other models. We screened over 160 drugs in ETMR-FBO and identified antracyclines and Triptolide as having anti-tumor, yet non-neurotoxic effects. Our in-depth transcriptomic analysis identified new therapeutic targets, including PDGFR-signaling for ETMR. Building on this insight, we disrupted tumor-TME interactions and observed significant anti-tumor effects both in vitro and in vivo. CONCLUSIONS EBT-FBO provide embryonal brain tumors like ETMR or AT/RT with their intrinsic, immature neuronal microenvironment essential for recapitulating natural tumor heterogeneity. This marks a significant advancement towards targeted therapy and patient-specific models in precision medicine.

OTHR-12. GENERATING A COMPREHENSIVE INTERACTIVE PEDIATRIC BRAIN TUMOR VISUALIZATION PORTAL FROM MOLECULAR AND CLINICAL DATA DERIVED FROM TWO MULTI-INSTITUTIONAL PROTOCOLS (SJMB03, SJYC07) AND NON-PROTOCOL PATIENTS TREATED AT ST JUDE CHILDREN’S RESEARCH HOSPITAL

Abstract BACKGROUND Pediatric central nervous system (CNS) tumors represent a diverse group of neoplasms with heterogeneous molecular and clinical characteristics. The need for well-organized, searchable datasets is paramount to enhance our understanding and improve the treatment approaches for these devastating diseases. To illustrate this, we have organized multifaceted data from patients into an interactive portal equipped with cutting-edge visualization tools. METHODS Clinical and molecular data from the SJMB03 and SJYC07 protocols and select published non-protocol St. Jude patients were collated and harmonized. A data dictionary was constructed to record demographic information, treatment details, histology, methylation classification, mutations, chromosomal aberrations, and outcomes. We implemented five visualization techniques - summary plots, sample view, scatter plots, sample matrix, and survival plots. These techniques are interconnected, enabling interaction and “crosstalk”. RESULTS Data from 710 patients with CNS tumors were incorporated: 386 Medulloblastomas (MB); 88 Atypical teratoid Rhabdoid Tumors (ATRT); 54 Ependymomas (EPN); 49 Pineoblastomas (PB); 28 Embryonal Tumors with Multilayered Rosettes (ETMR), 22 Infant-type hemispheric glioma (IHG), 16 High grade glioma (HGG), 13 Choroid Plexus Tumors (CPT); 11 CNS Sarcoma; 8 CNS tumor with BCOR internal tandem duplication (CNS BCOR); 6 CNS neuroblastoma FOXR2 activated (CNS NB FOXR2); 5 Low grade glioma (LGG); and 24 other entities or unclassified tumors. The portal features enabled the creation of custom cohorts. For example, patients with TP53 mutations (n=32; 15 MB, 7 CPT, 6 HGG, 1 CNS Sarcoma, 1 CNS NB FOXR2, 1 PB, 1 unclassified tumor) or DICER1 mutations (n=10; 7 PB, 2 CNS Sarcoma, and 1 ETMR) could be identified, cross-compared and analyzed adjusting for variables at users’ choice. CONCLUSION The integration of molecular and clinical data from multiple sources into an interactive visualization portal presents a comprehensive resource for research and clinical practice. Future work will focus on expanding the portal to integrate more cases/cohorts.

ETMR-15. RECLASSIFICATION OF CHILDHOOD CENTRAL NERVOUS SYSTEM PRIMITIVE NEUROECTODERMAL TUMOR: CLINICAL FEATURES AND OUTCOME

Abstract BACKGROUND The “central nervous system primitive neuroectodermal tumor” (CNS-PNET) nosology was removed from the World Health Organization (WHO) classification after DNA methylation profiling and new tumor entities characterized by specific recurrent genetic alterations were described. The aim of this study was to re-evaluate the diagnosis of CNS-PNET and to describe their clinical characteristics. METHODS Patients aged < 18 years with CNS-PNET or unclassified tumor, who were treated at Gustave Roussy between 1999 and 2019 or enrolled in the PNET HR trial were retrospectively identified. The study included all patients with sufficient archival material for diagnosis re-evaluation. RESULTS Sixty-two patients were eligible for the current analysis. The central histopathological review proposed a diagnosis according to the WHO classification in 58 cases. Out of these cases, 34 embryonal tumors were identified: 22 Embryonal Tumors with Multilayered Rosettes (ETMR), 4 CNS neuroblastomas with FOXR2 activation (CNS NB-FOXR2), 4 CNS high-grade neuroepithelial tumors with BCOR alteration (CNS HGNET-BCOR), 4 medulloblastomas. Another 24 tumors were classified as non embryonal tumors: 11 high-grade gliomas, 5 ependymomas, 4 pineoblastomas, 2 teratomas, one desmoplastic small round cell tumor, and one pilocytic astrocytoma. Four tumors without suspected diagnosis after first analysis remain unclassified after DNA methylation profiling. The 5-year event-free survival and overall survival for patients with ETMR were 14% (95% CI, 4.7-39%) and 27% (95% CI, 14-54%) respectively. For patients with CNS HGNET-BCOR and CNS NB-FOXR2 the median time to relapse was respectively 11 months (range, 0.5 to 1.3 year) and 3.1 years (range 1.7 to 4.4 years). CONCLUSIONS This study confirmed that the former nosology of CNS PNET does not correspond to a homogenous class but encompasses a diversity of rare histomolecular entities with specific clinical characteristics and outcomes.

ETMR-20. CARDIOLIPIN ACYL CHAIN MODULATION AS A NOVEL MITOCHONDRIAL-FOCUSED THERAPEUTIC TARGET IN ETMR

Abstract Embryonal tumor with multilayered rosettes (ETMR) is a highly aggressive CNS neoplasm that occurs almost exclusively in infants and is associated with an extremely poor prognosis. Dysregulated mitochondrial bioenergetics and dynamics have been associated with the initiation and progression of diverse cancers and studies have linked metabolic rewiring to chemotherapy resistance. Cardiolipins are mitochondrial-specific lipids that reside in the mitochondrial membrane and their fatty acid composition has been extensively shown to regulate mitochondrial function and dynamics. Despite the known functional significance of cardiolipin structure, their role in mitochondrial regulation of brain tumors remains ill-defined. Using mass spectrometry imaging, we identified a shift to shorter acyl chain cardiolipins within the rapidly proliferating embryonal tumor cells in patient samples and patient-derived cell lines grown as 3D tumorspheres. Western blot analysis of the enzymes involved in cardiolipin synthesis and remodeling identified a significant increase in the expression of the cardiolipin remodeling enzyme, LCLAT1/ALCAT1. Orthogonal imaging techniques including immunohistochemistry, transmission electron microscopy and super-resolution microscopy correlated shorter acyl chain remodeling of cardiolipin with fragmented mitochondria (increased fission) and aberrant cristae structure. Further studies identified increased expression of the fission protein Drp1, decreased expression of respiratory chain enzymes and altered respiration in the embryonal tumor cells. Ongoing studies will utilize multiple methods to modulate cardiolipin acyl chain structure and use MS and MSI to correlate cardiolipin fatty acid structure to mitochondrial function and growth inhibition in the embryonal tumor cells.

Genetic mutation and immune infiltration in embryonal tumor with multilayered rosettes.

Genetic mutations stand as pivotal factors leading to the occurrence of embryonal tumor with multilayered rosettes (ETMR). This study aims to identify improved treatment approaches by unraveling the genetic drivers and immune infiltration in ETMR. Two siblings with ETMR, treated at the General Hospital of Ningxia Medical University, were enrolled. Diagnosis involved MRI, Hematoxylin and Eosin (HE), and immunohistochemical (IHC) staining. Differentially expressed genes (DEGs) in ETMR were identified using GSE122077 and GSE14296 datasets. GO and KEGG analyses were used to determine ETMR-related pathways. Whole exome sequencing (WES) was utilized to annotate genetic variations in ETMR. Core genes, identified by protein-protein interaction (PPI), formed a diagnostic model evaluated by Logistic Regression. Single-sample Gene Set Enrichment Analysis (ssGSEA) assessed immune infiltration in ETMR, examining correlations between immune cells and core genes. Two siblings were diagnosed with ETMR. In ETMR, 135 DEGs were identified, of which 25 genes were annotated with 28 mutation sites. Moreover, ETMR-related pathways included cell cycle, synaptic functions, and neurodegeneration. Three ETMR-related core genes (ALB, PSMD1, and PAK2) were screened by protein-protein interaction (PPI). The diagnostic model constructed using these genes demonstrated an AUC value of 0.901 (95% CI: 0.811-0.991) in the training set, indicating accurate predictions in ETMR. Enhanced ssGSEA scores for 16 immune cells in ETMR tissues suggested a strong immune response. This study identifies diagnostic models associated with three core variant genes (ALB, PSMD1, PAK2) and enhanced immune cell activity in ETMR. It reveals crucial genetic features and significant immune responses in ETMR.

Integrated proteomics spotlight the proteasome as a therapeutic vulnerability in embryonal tumors with multilayered rosettes.

Embryonal tumors with multilayered rosettes (ETMR) are rare malignant embryonal brain tumors. The prognosis of ETMR is poor and novel therapeutic approaches are desperately needed. Comprehension of ETMR tumor biology is currently based on only few previous molecular studies, which mainly focused on the analyses of nucleic acids. In this study, we explored integrated ETMR proteomics. Using mass spectrometry, proteome data were acquired from 16 ETMR and the ETMR cell line BT183. Proteome data were integrated with case-matched global DNA methylation data, publicly available transcriptome data, and proteome data of further embryonal and pediatric brain tumors. Proteome-based cluster analyses grouped ETMR samples according to histomorphology, separating neuropil-rich tumors with neuronal signatures from primitive tumors with signatures relating to stemness and chromosome organization. Integrated proteomics showcased that ETMR and BT183 cells harbor proteasome regulatory proteins in abundance, implicating their strong dependency on the proteasome machinery to safeguard proteostasis. Indeed, in vitro assays using BT183 highlighted that ETMR tumor cells are highly vulnerable toward treatment with the CNS penetrant proteasome inhibitor Marizomib. In summary, histomorphology stipulates the proteome signatures of ETMR, and proteasome regulatory proteins are pervasively abundant in these tumors. As validated in vitro, proteasome inhibition poses a promising therapeutic option in ETMR.

Outcomes Following Radiation Therapy (RT) for Very Young Age CNS Embryonal Tumors on COG ACNS0334 According to Molecular-Confirmed Diagnosis

The outcomes of upfront or relapse radiation therapy (RT) for the Children's Oncology Group ACNS0334 protocol based on molecular diagnosis were assessed. Therapy included maximal safe surgery, high-dose chemotherapy with stem cell rescue, randomization for inclusion of high dose methotrexate (MTX) and optional RT. There were 24 patients that received RT on COG ACNS0334 of 77 evaluable patients with a diagnosis of either high-risk medulloblastoma (MB) or supratentorial primitive neuroectodermal tumor (SPNET). RT was a recommendation (M0: Focal, M+: CSI 18 Gy) given young patient age <36 months at enrollment. Seven RT patients were excluded for ineligible pathology (1 ATRT, 1 HGG) or insufficient tissue. The aim of this report is to review outcomes of 17 patients on ACNS0334 receiving RT (8 Upfront, 9 at relapse) with a molecular diagnosis that included MB, Pineoblastoma (PB), or Embryonal tumor with multilayered rosettes (ETMR). In the MB group, there were 9 patients irradiated with MB (Group 3 = 8, SHH = 1). 5-year OS for MB Group 3 receiving RT (median primary dose 54 Gy) was 62.5% with no difference observed comparing 6 patients treated with upfront RT versus 2 treated at relapse (p = 0.27). All upfront RT for MB Group 3 had initial partial response (PR) to 0334 chemotherapy. RT delivery for upfront RT MB Group 3 included craniospinal radiation (CSI) in 5 patients and 1 patient who received focal RT to the primary (50 Gy) and metastatic site (44 Gy). Eighty percent of CSI for upfront RT in Group 3 was 18 Gy or 23.4 Gy. Relapse RT for MB Group 3 (2 patients) utilized full dose CSI (36 Gy, 39.6 Gy) and both patients are survivors with 5+ years follow-up. CSI dose for Group 3 MB was higher for relapse RT (mean 37.8 Gy) as compared to upfront RT (mean 19.8 Gy, p = 0.013). Use of MTX was 50% in both upfront RT and relapse RT Group 3 MB cohorts. One patient with MB SHH (classic histology) underwent upfront focal RT (54 Gy) after initial PR to systemic therapy (without MTX) and is surviving 5+ years. PB: Of 4 PB patients (median primary dose 48.8 Gy) 1 had RT upfront (CSI 18 Gy) and 3 had RT at relapse (1 patient received CSI, 21 Gy). All patients with PB expired within 2 years. MTX was given in 75% (including 1 upfront RT PB). Two of 3 patients treated at relapse had prior complete response (CR). ETMR: All 4 patients (median primary dose 54 Gy) with ETMR were treated at relapse, with CSI given in 1 patient (23.4 Gy). All patients with ETMR expired within 2 years, and 2 (50%) had received MTX. Two patients (50%) had initial CR. The RT cohort for Group 3 MB on ACNS0334 exhibited long-term survival both for both upfront and relapse RT, however relapsed Group 3 MB received higher dose CSI. RT upfront for MB, including one surviving MB SHH patient receiving focal RT, was solely given for incomplete initial chemotherapy response. There were no survivors for either PB or ETMR when the majority (88%) were treated at relapse.

Medulloblastomas, CNS embryonal tumors, and cerebellar mutism syndrome: advances in care and future directions.

Central nervous system (CNS) embryonal tumors, commonly found in pediatric patients, represent a heterogeneous mix of lesions with an overall poor (though improving) prognosis. Medulloblastomas are by far the most frequently encountered and most widely studied subtype, though others include atypical teratoid/rhabdoid tumors (AT/RTs), embryonal tumor with multilayered rosettes (ETMRs), and CNS neuroblastomas, FOX-R2 activated. The classification, diagnosis, and treatment of these lesions have evolved drastically over the years as their molecular underpinnings have been elucidated. We describe the most recent 2021 WHO Classification system, discuss current understanding of the genetic basis, and demonstrate current thinking in treatment for these highly complex tumors. Since surgical resection continues to remain a mainstay of treatment, preventing and managing surgical complications, especially cerebellar mutism syndrome (CMS), is paramount. We describe the current theories for the etiology of CMS and two centers' experience in mitigating its risks. As our surgical toolbox continues to evolve along with our understanding of these tumors, we hope future patients can benefit from both improved overall survival and quality of life.

Abstract 4875: The anti-tumor effects of GSK-3β inhibitor (9-1NG-41) in ETMR and ATRT pediatric brain tumors

Abstract Purpose Embryonal brain tumors (EBTs): ETMR (embryonal tumor with multilayered rosettes) and ATRT (atypical teratoid rhabdoid tumor) are aggressive malignancies with poor prognosis necessitating alternative strategies. GSK-3β is a serine threonine kinase often overexpressed in solid tumors and a positive regulator of NF-κB which promotes tumor growth and chemotherapy resistance. 9-ING-41 is a maleimide-based small molecule that crosses the blood brain barrier and selectively inhibits GSK-3β. Previously we demonstrated that 9-ING-41 decreases ETMR and ATRT cell viability through apoptosis and increases p53 signaling, however the exact mechanism leading to its activation is unknown. This study aims to determine the optimal dosing for 9-ING-41 and describe the phenotypic response of these cell lines to 9-ING-41 using cell viability assay, western blotting and neurosphere assay. This information will help further elucidate the mechanism of action for this drug. Methods ATRT cell lines ATRT 2141, ATRT-787199, ATRT 803499, LCH-091-07 and the ETMR cell line BT-183 were grown for 24hrs prior to 9-ING-41 treatment in DMEM and Neurocult medium, respectively. Cell viability was assessed after 72hrs of treatment using Cell Titer Glo 2.0. Western blots were analyzed for XIAP, cleaved caspase-3, p53, MDM2 after 24 and 48 hours at 200nM, 400nM and 600nM. Neurosphere assays were done by seeding two cells per well in a 96-well plate followed by treatment with 9-ING-41. Neurosphere frequency and size was monitored weekly for up to 4 weeks using Incucyte® S3 software. Pearson’s correlation graph was generated to determine the relationship between GSK-3β expression and sensitivity to 9-ING-41. Graph Pad Prism was used for statistical analysis and level of significance set to p=0.05. Results The IC50s of 9-ING-41 in cell lines ATRT 2141, ATRT-787199, ATRT 803499 and LCH-091-07 were 194nM, 558nM, 730nM &amp; 5353nM, respectively. We observed decreased expression of NF-κB mediated anti-apoptotic marker XIAP and an increase in expression of apoptotic marker cleaved caspase-3 and tumor suppressor p53 in response to 9-ING-41. MDM2, a negative regulator of p53 had a significant increase in expression indicating it is not responsible for p53 increase which may suggest ATM pathway could be responsible for regulating p53 increase. GSK-3β expression in vitro, correlated with sensitivity to 9-ING-41. There was a dose dependent decrease in frequency and size of neurospheres in response to 9-ING-41. Conclusion 9-ING-41 decreases cell viability and neurosphere formation in ETMR and ATRT cell lines within clinically relevant doses. p53 stabilization is not mediated by MDM2 and may be downstream of the ATM pathway. Further studies are underway to demonstrate its efficacy in mouse models. Citation Format: Divya Gandra, Lauren Difabio, Kaitlyn H. Smith, Kimberly Q. McKinney, Giselle S. Sholler. The anti-tumor effects of GSK-3β inhibitor (9-1NG-41) in ETMR and ATRT pediatric brain tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4875.

Embryonal tumor with multilayered rosettes: Post‐treatment maturation and implications for future therapy

Embryonal tumor with multilayered rosettes (ETMR) is a deadly grade IV pediatric brain tumor. Despite an intensive multimodal treatment approach that includes surgical resection, high-dose chemotherapy, and radiotherapy, the progression-free survival at 5 years is less than 30%. We report a case of long-term survival in a 5-month old female with a large mass in the posterior fossa, diagnosed as ETMR, which subsequently underwent treatment-induced maturation. Prior to chemotherapy, histopathology revealed an abundance of highly proliferative, undifferentiated cells and multilayered rosette structures. Conversely, post-treatment histopathology revealed cell populations that differentiated into neuronal and ganglionic phenotypes. At 5-year follow-up, the patient remains progression-free. This finding contributes to the few reports to date of post-treatment differentiation/maturation of ETMR cell populations, with an implication for less cytotoxic therapeutic interventions aimed at differentiation.

Imaging findings of supratentorial embryonal tumor with multilayered rosettes in children

We analyzed and summarized the imaging characteristics and clinical data of seven pediatric supratentorial embryonal tumors with multilayered rosettes (ETMR). There were four boys and three girls aged from two to six years old. Pediatric supratentorial ETMR often presented large cystic and solid mass, calcification, significant mass effect and mild peritumoral edema. The solid part often showed heterogeneous mild enhancement. In combination with the location of tumor and age of onset, the typical imaging manifestations of supratentorial ETMR in children are valuable for accurate diagnosis.

Clinical Management of Embryonal Tumor with Multilayered Rosettes: The CCMC Experience.

Objective: Embryonal tumors with multilayered rosettes (ETMRs) are highly aggressive pediatric brain tumors with poor prognosis. No standard treatment strategy for them exists because of their rarity. This study aimed to share experiences on the clinical diagnosis and treatment of ETMRs at China Children’s Medical Center (CCMC). Methods: Patients who received a diagnosis of an ETMR between January 2017 and June 2020 were included. Clinical characteristics, such as age of onset, tumor size, stage, tumor site, treatment strategy, and clinical outcome, were retrospectively analyzed. Results: There were four boys and one girl within 4 years who received a diagnosis during this 4-year timeframe, and were thus included. The average age of morbidity was 29 months (range 16–66 months). The common clinical presentation was headaches and nausea caused by intracranial hypertension. All four patients were chromosome 19 microRNA cluster (C19MC) amplification positive. Two patients achieved complete remission, and one patient attained partial remission after multimodal treatment. Of the two deaths, one died from the rapid progression of the disease and another from tumor-related complications. Conclusion: ETMRs are extremely rare brain tumors with a high, early mortality in children. Surgery is the mainstream treatment for ETMRs. Some patients may also benefit from postoperative adjuvant chemotherapy and radiotherapy.