RELA Fusion Research Articles

ANGI-01. EXTRANEURAL METASTATIC EPENDYMOMA: DISTANT METASTASIS TO THE PLEURA, LUNGS, LYMPH NODES, AND BONE

Abstract Ependymomas are neuroepithelial tumors arising from ependymal cells surrounding the cerebral ventricles that account for 1.6-1.8% of primary CNS tumors. They rarely metastasize to extraneural structures, with one study reporting an incidence of 6.2% with a mean time from diagnosis to extraneural metastasis of 75.6 months. This spread, when it does occur, has been reported to occur to the lungs, lymph nodes, liver, and bone. We describe the case of a patient with recurrent WHO Grade III ependymoma, ZFTA::RELA fusion positive, with extraneural metastatic disease to the thoracic spine, lungs, and pelvic bones. He was treated with multiple surgical resections, radiation therapy, and salvage chemotherapy for his extraneural metastasis to the lungs, bone, pleural space, and lymph nodes. His functional and respiratory statuses declined until he ultimately developed pneumonia, status epilepticus, then spontaneous respiratory arrest, which resulted in his passing away. Salvage chemotherapy for ependymoma has unclear mortality benefit and more research must be done to determine how best to treat patients with extraneural metastases, particularly those who fail traditional treatment with resection and radiation therapy.

Novel case of ependymoma-like tumor with mesenchymal differentiation harboring ZFTA::RELA fusion in an adult.

High-grade supratentorial tumors harboring ZFTA::NCOA1/2 fusion in infants presenting with mixed histology of embryonal-appearing components resembling ependymoma and mesenchymal sarcomatous components have recently been reported as ependymoma-like tumors with mesenchymal differentiation (ELTMDs). In contrast, we describe herein a pathologically similar case with a novel ZFTA::RELA fusion in an adult. A frontal lobe lesion was resected from a 30-year-old woman and displayed mixed components on pathological examination, showing ependymoma-like and sarcomatous parts. The absence of perivascular pseudorosettes was inconsistent with a diagnosis of ependymoma. Fluorescence in situ hybridization analysis confirmed ZFTA::RELA fusion. The DKFZ methylation classifier (v12.8) did not categorize this case among established methylation classes. In addition, t-distributed stochastic neighbor embedding analysis using DNA methylation data revealed that the present case was distant from ependymomas but close to two previously reported cases of ELTMD involving ZFTA::NCOA1/2 fusion. Taken together, we concluded that this tumor should be considered under the entity of ELTMD. This represents the first description of an adult patient with ELTMD harboring ZFTA::RELA fusion analyzed by DNA methylation profiling, supporting the establishment of ELTMD as a possible new tumor type.

MODL-08. PATIENT-DERIVED ORGANOIDS RECAPITULATE THE MOLECULAR LANDSCAPE OF PEDIATRIC BRAIN TUMORS

Abstract BACKGROUND Pediatric brain tumors (PBT) are the leading cause of non-accidental death in pediatric age. These tumors are rare and lack adequate experimental models capable of accurately capturing their complex molecular landscape. METHODS AND RESULTS Our team has established specific protocols for the generation and expansion of ex-vivo patient-derived organoids (PDO) from fresh surgical material of a variety of PBT, within a clinically relevant timeframe. These include pilocytic astrocytomas, a pleomorphic xanthoastrocytoma, a rosette-forming glioneuronal tumor, a supratentorial ependymoma, diffuse high-grade gliomas, a medulloblastoma, an atypical teratoid rhabdoid tumor, and a meningioma. The PDO cultures were set within 7-15 days, with organoids reaching 200-800 μm in diameter. Immunofluorescence analysis using confocal high-content microscopy imaging revealed that PDO kept the protein markers typically found in the corresponding primary tumors. Likewise, at a genomic level, PDO recapitulated the molecular landscape of corresponding primary tumors, as denoted by the maintenance of NF1, PIK3CA, and FGFR1mutations (rosette-forming glioneuronal tumor), KIAA1549::BRAF fusions (pilocytic astrocytomas), ZFTA::RELA fusion(supratentorial ependymoma) or gene amplifications, such as KRAS, CDK4, MDM2, GLI1, PTPN11 and PI3KCA. Additionally, PDO exhibited minimal genetic drift after over one month in culture. In three PBT cases from a medulloblastoma, an atypical teratoid rhabdoid tumor, and a meningioma, where no molecular alterations were detected using a comprehensive NGS panel, we are running DNA methylation arrays and copy number variations. CONCLUSIONS Our results underscore the potential of PDO as a robust ex-vivo experimental model. The generated PDO cultures can be rapidly established and retain key molecular features of corresponding primary tumors. This preclinical model presents versatile applications, serving as a valuable molecular tool for precision medicine approaches in PBT patients, the study of PBT biology, and the acceleration of drug development processes within the context of PBT.

An In-Depth Analysis of Ependymoma: Neuropathological Insights, Therapeutic Strategies, and Prognostic Implications in Intracranial Ependymal Tumors

Ependymomas are primary central nervous system (CNS) neoplasms arising from ependymal cells lining the ventricular system and spinal cord. They represent a heterogeneous group of tumors with distinct histological and molecular subtypes, making their diagnosis, management, and prognosis a complex and evolving challenge in the field of neuro-oncology. This comprehensive review delves into the intricate landscape of ependymomas, elucidating their pathogenesis, classification, and molecular underpinnings. Histologically, they can manifest as myxopapillary, subependymoma, and anaplastic ependymomas, each carrying distinct clinical implications and therapeutic strategies. Moreover, recent advancements in molecular profiling have unveiled significant genetic alterations such as amplifications in RELA, YAP1, and C11orf95-MAML2 fusion, which have profound implications for prognosis and therapeutic decision-making. We explore the intricacies of clinical management, encompassing surgical resection, radiation therapy, and chemotherapy regimens tailored to the tumor's location, grade, and molecular profile. Despite their location within the CNS, ependymomas are notorious for their diverse clinical presentations, including headache, neurological deficits, and intracranial hypertension, necessitating multidisciplinary care and vigilant surveillance. Additionally, this article investigates the latest developments in targeted therapies, immunotherapeutic approaches, and ongoing clinical trials in an attempt to provide a glimpse into the future of ependymoma treatment, as precision medicine begins to play a more significant role in guiding therapeutic decisions. Furthermore, the abstract examines the critical issue of ependymoma prognosis, emphasizing the importance of integrating histopathological and molecular data in order to stratify patients into risk categories more accurately. The role of genetic markers, such as chromosome 1q gain and chromosome 6q loss, in predicting outcomes is thoroughly explored. In summary, this article aims to offer a comprehensive perspective on ependymomas, from their cellular origins to the latest breakthroughs in their management, offering insights into their clinical course, and underscoring the critical role of multidisciplinary collaboration in advancing the understanding and treatment of these complex intracranial tumors.

Supratentorial cortical ependymoma: A systematic literature review and case illustration.

Cortical ependymomas are currently not considered a subgroup of supratentorial ependymomas; however, there is a growing body of literature investigating the natural history of these lesions compared to supratentorial ependymomas. We performed a systematic literature review of cortical ependymomas with a focus on the natural history, clinical characteristics, and clinical outcomes of these lesions as compared to supratentorial ependymomas. Our search revealed 153 unique cases of cortical ependymomas. The mean age on presentation was 21.2 years. Males and females comprised 58.8% (90/153) and 41.2% (63/153) of cases, respectively. The most common presenting symptom was seizure activity occurring in 44.4% of the cohort (68/153). The recently recognized C11orf95-RELA fusion was identified in 13.7% of the cohort (21/153) and 95.5% of cases (21/22) reporting molecular characterization. World Health Organization grades 2 and 3 were reported in 52.3% (79/151) and 47.7% (72/151) of cases, respectively. The frontal lobe was involved in the majority of cases (54.9%, 84/153). Gross total resection was achieved in 80.4% of cases (123/153). Tumor recurrence was identified in 27.7% of cases (39/141). Mean clinical follow-up was 41.3 months. Mean overall survival of patients who expired was 27.4 months whereas mean progression-free survival was 15.0 months. Comparatively, cortical ependymomas with C11orf95-RELA fusions and supratentorial ependymomas with C11orf95 RELA fusions exhibited differing clinical outcomes. Further studies with larger sample sizes are necessary to investigate the significance of RELA fusions on survival in cortical ependymomas and to determine whether cortical ependymomas with C11orf95-RELA fusions should be classified as a distinct entity.

Activation of Hedgehog signaling by the oncogenic RELA fusion reveals a primary cilia-dependent vulnerability in supratentorial ependymoma.

Supratentorial RELA fusion (ST-RELA) ependymomas (EPNs) are resistant tumors without an approved chemotherapeutic treatment. Unfortunately, the molecular mechanisms that lead to chemoresistance traits of ST-RELA remain elusive. The aim of this study was to assess RELA fusion-dependent signaling modules, specifically the role of the Hedgehog (Hh) pathway as a novel targetable vulnerability in ST-RELA. Gene expression was analyzed in EPN from patient cohorts, by microarray, RNA-seq, qRT-PCR, and scRNA-seq. Inhibitors against Smoothened (SMO) (Sonidegib) and Aurora kinase A (AURKA) (Alisertib) were evaluated. Protein expression, primary cilia formation, and drug effects were assessed by immunoblot, immunofluorescence, and immunohistochemistry. Hh components were selectively overexpressed in EPNs induced by the RELA fusion. Single-cell analysis showed that the Hh signature was primarily confined to undifferentiated, stem-like cell subpopulations. Sonidegib exhibited potent growth-inhibitory effects on ST-RELA cells, suggesting a key role in active Hh signaling; importantly, the effect of Sonidegib was reversed by primary cilia loss. We, thus, tested the effect of AURKA inhibition by Alisertib, to induce cilia stabilization/reassembly. Strikingly, Alisertib rescued ciliogenesis and synergized with Sonidegib in killing ST-RELA cells. Using a xenograft model, we show that cilia loss is a mechanism for acquiring resistance to the inhibitory effect of Sonidegib. However, Alisertib fails to rescue cilia and highlights the need for other strategies to promote cilia reassembly, for treating ST-RELA tumors. Our study reveals a crucial role for the Hh pathway in ST-RELA tumor growth, and suggests that rescue of primary cilia represents a vulnerability of the ST-RELA EPNs.

In This Issue

In This Issue

Supratentorial non-RELA, ZFTA-fused ependymomas: a comprehensive phenotype genotype correlation highlighting the number of zinc fingers in ZFTA-NCOA1/2 fusions

The cIMPACT-NOW Update 7 has replaced the WHO nosology of “ependymoma, RELA fusion positive” by “Supratentorial-ependymoma, C11orf95-fusion positive”. This modification reinforces the idea that supratentorial-ependymomas exhibiting fusion that implicates the C11orf95 (now called ZFTA) gene with or without the RELA gene, represent the same histomolecular entity. A hot off the press molecular study has identified distinct clusters of the DNA methylation class of ZFTA fusion-positive tumors. Interestingly, clusters 2 and 4 comprised tumors of different morphologies, with various ZFTA fusions without involvement of RELA. In this paper, we present a detailed series of thirteen cases of non-RELA ZFTA-fused supratentorial tumors with extensive clinical, radiological, histopathological, immunohistochemical, genetic and epigenetic (DNA methylation profiling) characterization. Contrary to the age of onset and MRI aspects similar to RELA fusion-positive EPN, we noted significant histopathological heterogeneity (pleomorphic xanthoastrocytoma-like, astroblastoma-like, ependymoma-like, and even sarcoma-like patterns) in this cohort. Immunophenotypically, these NFκB immunonegative tumors expressed GFAP variably, but EMA constantly and L1CAM frequently. Different gene partners were fused with ZFTA: NCOA1/2, MAML2 and for the first time MN1. These tumors had epigenetic homologies within the DNA methylation class of ependymomas-RELA and were classified as satellite clusters 2 and 4. Cluster 2 (n = 9) corresponded to tumors with classic ependymal histological features (n = 4) but also had astroblastic features (n = 5). Various types of ZFTA fusions were associated with cluster 2, but as in the original report, ZFTA:MAML2 fusion was frequent. Cluster 4 was enriched with sarcoma-like tumors. Moreover, we reported a novel anatomy of three ZFTA:NCOA1/2 fusions with only 1 ZFTA zinc finger domain in the putative fusion protein, whereas all previously reported non-RELA ZFTA fusions have 4 ZFTA zinc fingers. All three cases presented a sarcoma-like morphology. This genotype/phenotype association requires further studies for confirmation. Our series is the first to extensively characterize this new subset of supratentorial ZFTA-fused ependymomas and highlights the usefulness of ZFTA FISH analysis to confirm the existence of a rearrangement without RELA abnormality.

A coordinated approach for the assessment of molecular subgroups in pediatric ependymomas using low-cost methods

Although ependymoma (EPN) molecular subgroups have been well established by integrated high-throughput platforms, low- and middle-income countries still need low-cost techniques to promptly classify these molecular subtypes. Here, we applied low-cost methods to classify EPNs from a Brazilian cohort with 60 pediatric EPN patients. Fusion transcripts (C11orf95-RELA, YAP1-MAMLD1, and YAP1-FAM118B) were investigated in supratentorial EPN (ST-EPNs) samples through RT-PCR/Sanger sequencing and immunohistochemistry (IHC) for p65/L1CAM. qRT-PCR and IHC were used to evaluate expression profiling of CXorf67, LAMA2, NELL2, and H3K27me3 in posterior fossa EPN (PF-EPNs) samples. In silico analysis was performed using public microarray data to validate the molecular assignment PF-EPNs with LAMA2/NELL2 markers. RELA cases and YAP1-MAMLD1 fusions were identified in nine and four ST-EPNs, respectively. An additional RELA case was identified by IHC. Of note, LAMA2 and NELL2 gene expression and immunoprofiling were less accurate for classifying PF-EPNs, which were confirmed by in silico analysis. Yet, H3K27me3 staining was sufficient to classify PF-EPN subgroups. Our results emphasize the feasibility of a simplified strategy to molecularly classify EPNs in the vast majority of cases (49/60; 81.7%). A coordinated combination of simple methods can be effective to screen pediatric EPN with the available laboratory resources at most low-/mid-income countries, giving support for clinical practice in pediatric EPN. KEY MESSAGES: Low- and middle-income countries need effective low-cost approaches to promptly distinguish between EPN molecular subgroups. RT-PCR plus Sanger sequencing is able to recognize the most common types of RELA and YAP1 fusion transcripts in ST-EPNs. Genetic and protein expressions of LAMA2 and NELL2 are of limited value to accurately stratify PF-EPNs. Immunohistochemical staining for H3K27me3 may be used as a robust method to accurately diagnose PF-EPNs subgroups. A coordinated flow diagram based on these validated low-cost methods is proposed to help clinical-decision making and to reduce costs with NGS assessment outside research protocols.

Cross-Species Genomics Reveals Oncogenic Dependencies in ZFTA/C11orf95 Fusion-Positive Supratentorial Ependymomas.

Molecular groups of supratentorial ependymomas comprise tumors with ZFTA-RELA or YAP1-involving fusions and fusion-negative subependymoma. However, occasionally supratentorial ependymomas cannot be readily assigned to any of these groups due to lack of detection of a typical fusion and/or ambiguous DNA methylation-based classification. An unbiased approach with a cohort of unprecedented size revealed distinct methylation clusters composed of tumors with ependymal but also various other histologic features containing alternative translocations that shared ZFTA as a partner gene. Somatic overexpression of ZFTA-associated fusion genes in the developing cerebral cortex is capable of inducing tumor formation in vivo, and cross-species comparative analyses identified GLI2 as a key downstream regulator of tumorigenesis in all tumors. Targeting GLI2 with arsenic trioxide caused extended survival of tumor-bearing animals, indicating a potential therapeutic vulnerability in ZFTA fusion-positive tumors. SIGNIFICANCE: ZFTA-RELA fusions are a hallmark feature of supratentorial ependymoma. We find that ZFTA acts as a partner for alternative transcriptional activators in oncogenic fusions of supratentorial tumors with various histologic characteristics. Establishing representative mouse models, we identify potential therapeutic targets shared by ZFTA fusion-positive tumors, such as GLI2.This article is highlighted in the In This Issue feature, p. 2113.

Ependymoma-like tumor with mesenchymal differentiation harboring C11orf95-NCOA1/2 or -RELA fusion: A hitherto unclassified tumor related to ependymoma.

Recurrent fusion genes involving C11orf95, C11orf95‐RELA, have been identified only in supratentorial ependymomas among primary CNS tumors. Here, we report hitherto histopathologically unclassifiable high‐grade tumors, under the tentative label of “ependymoma‐like tumors with mesenchymal differentiation (ELTMDs),” harboring C11orf95‐NCOA1/2 or ‐RELA fusion. We examined the clinicopathological and molecular features in five cases of ELTMDs. Except for one adult case (50 years old), all cases were in children ranging from 1 to 2.5 years old. All patients presented with a mass lesion in the cerebral hemisphere. Histologically, all cases demonstrated a similar histology with a mixture of components. The major components were embryonal‐appearing components forming well‐delineated tumor cell nests composed of small uniform cells with high proliferative activity, and spindle‐cell mesenchymal components with a low‐ to high‐grade sarcoma‐like appearance. The embryonal‐appearing components exhibited minimal ependymal differentiation including a characteristic EMA positivity and tubular structures, but histologically did not fit with ependymoma because they lacked perivascular pseudorosettes, a histological hallmark of ependymoma, formed well‐delineated nests, and had diffuse and strong staining for CAM5.2. Molecular analysis identified C11orf95‐NCOA1, ‐NCOA2, and ‐RELA in two, one, and two cases, respectively. t‐distributed stochastic neighbor embedding analysis of DNA methylation data from two cases with C11orf95‐NCOA1 or ‐NCOA2 and a reference set of 380 CNS tumors revealed that these two cases were clustered together and were distinct from all subgroups of ependymomas. In conclusion, although ELTMDs exhibited morphological and genetic associations with supratentorial ependymoma with C11orf95‐RELA, they cannot be regarded as ependymoma. Further analyses of more cases are needed to clarify their differences and similarities.

Survival and Prognostic Factors of Adult Intracranial Ependymoma: A Single-institutional Analysis of 236 Patients.

Adult intracranial ependymomas (EPNs) are extremely rare brain tumors. Currently, clinical and molecular factors that could inform individualized treatment strategies are still lacking for EPNs in this age group. The aim of this study was to investigate potential prognostic indicators and rational therapeutic management in a large cohort of adult intracranial EPNs. Adult patients who underwent resection of World Health Organization (WHO) grade II or III intracranial EPNs were included. The demographic features, clinicopathologic manifestations, molecular subgroups, and outcomes were retrospectively analyzed. Overall survival and progression-free survival were calculated using the Kaplan-Meier analysis. Potential prognostic indicators were identified using multivariable Cox proportional hazards model. This cohort included 236 adult patients with a mean age of 36.2 years (range: 18 to 72 y) at diagnosis. The tumor location was supratentorial (ST) in 102 (43.2%) and infratentorial in 134 (56.8%). Pathologic analysis revealed 43.1% of ST-EPNs with RELA fusion and 88.1% of posterior fossa ependymomas (PF-EPNs) with positive H3K27me3 staining. Gross total removal was achieved in 169 cases (71.6%). During follow-up, 97 (41.1%) patients had disease progression and 39 (16.5%) died. Kaplan-Meier analysis showed that patients with H3K27me3-positive PF-EPN had excellent survival, whereas patients with RELA fusion-positive ST-EPN or H3K27me3-negative PF-EPN had poor prognosis (progression-free survival: P=1.3E-16, overall survival: P=2.5E-12). Multivariate analysis showed that molecular subgroup, extent of resection, and Ki-67 index were strong independent prognostic indicators. In conclusion, our study provides essential information on the prognostic prediction of adult intracranial EPNs that will assist in establishing appropriate risk stratification and individualized treatment strategies in future clinical trials.

LINC-29. IMPACT OF RELA FUSION ON OUTCOMES OF CHILDHOOD SUPRATENTORIAL EPENDYMOMAS (ST-EPEN)

BACKGROUNDEpendymomas are heterogenous group of tumours with variable clinical course and diverse molecular features. RELA fusion status has been reported to have prognostic impact in ST-EPEN. Our retrospective study analysed the prevalence and clinical impact of RELA fusion in childhood ST-EPEN at our centre. STUDYMETHODSFFPE tissues of all childhood ST-EPEN diagnosed during 2011–2017 were evaluated for RELA fusion 1/2 by RT-PCR. Children were treated as per guidelines by the Neuro-oncology multidisciplinary team. Outcomes were correlated with RELA fusion, histological features and immunohistochemical parameters(L1CAM expression and Mib-1 index). Only patients with therapy details were included.RESULTSA total of 37 patients(0–50 years) with ST-EPEN were included(median age-10.2 years; boy:girl ratio-1.4:1)for analysis. Histological grade II, II/III and III was seen in 4(11%),2(5%) and 31(84%) patients respectively. Mib-1 index was assessable in 33 patients of which, 9 patients (24%) had a Mib-1 index >20%. RELA fusion was detected in 13(35%)tumors. The 3-year and 5-year EFS/OS of the overall cohort was 64.2%/83.6% and 60.1%/73.1% respectively. The 3-year/5-year EFS of RELA-positive tumors was inferior compared to RELA-negative tumours (53.8%/36% v/s 62.6%/53.6%; p=0.391). The 3-year/5-year EFS of tumors expressing L1CAM versus negative-expression was comparable (61.1%/55%v/s59.8%/47.9%;p=0.44). Presence of Mib-1>20% correlated with inferior survival (5-year EFS:81.1%vs22.2%; p<0.01).CONCLUSIONSST-EPEN with RELA fusion had trend towards increased relapse/progression. High Mib-1 correlated with poor survival. RELA fusion status needs to be studied in a larger cohort prospectively to confirm its clinical impact.

TBIO-13. USE OF NEXT GENERATION SEQUENCING TO IDENTIFY MOVE DRIVERS OF CRYPTIC, CLINICALLY AGGRESSIVE BRAIN TUMORS

INTRODUCTIONNext generation sequencing (NGS) is an emerging technology which allows for in-depth analysis of pediatric brain tumors. NGS has particular use in the context of ambiguous or aggressive neoplasms, where it can be leveraged to discover novel drivers, inform pathologic classification, and direct targeted therapies.OBJECTIVEThe objective of this case series was to utilize NGS technology to illuminate the biology of aggressive brain tumors with ambiguous pathologic features and clinically aggressive behavior.METHODSFFPE tumor tissue and matched germline DNA were subjected to whole exome sequencing (WES). Data were analyzed according to the GATK pipeline.RESULTSThe first case is a 6-year-old male who presented with innumerable foci of leptomeningeal nodules throughout the neuroaxis. Original pathology was CNS embryonal tumor. WES identified loss of chromosome 1p and 16q with gain of 1q and amplification of MYC and OTX2 loci (cytogenetic aberrations characteristic of group 3 medulloblastoma) and a deleterious mutation in BCL7B, a known tumor suppressor gene. The second case is a 2-year-old female who presented with a parietal lobe mass diagnosed as high grade neuroepithelial tumor with C11orf95 translocation, but no RELA fusion. WES revealed loss of small region of chromosome 2p and mutations in IDH3G, TRAF2, and JMJD1C, suggesting novel targets for further study.CONCLUSIONSIn both cases, NGS studies were able to shed light on the underlying tumor biology and/or refine the pathologic diagnosis. These data underscore the utility of applying NGS technology to study the biology of pediatric brain tumors.

EPEN-44. EXTRACELLULAR VESICLES OF SUPRATENTORIAL EPENDYMOMA RELA MEDIATE INTERACTIONS WITH CELLS OF THE TUMOR MICROENVIRONMENT

Ependymal tumors (EPNs) account for ~10% of all pediatric brain tumors. Supratentorial EPN characterized by RELA fusions (ST-EPN-RELA) and posterior fossa EPN group A (PF-EPN-A) form the two most frequent molecular groups, both of which are associated with poor prognosis and for which only limited therapeutic options are available. Since pediatric EPNs have a relatively low mutational burden, identification and characterization of tumor-associated pathways and molecular processes is of critical importance to inform potential therapeutic targets. Previous transcriptional studies implicated aberrant vesicular pathways in ST-EPN-RELA, prompting further investigation into their putative role in EPN pathogenesis. To this aim, we isolated extracellular vesicles (EVs) of ST-EPN-RELA patient derived cell lines and performed protein mass spectrometry. The specific ST-EPN-RELA EV protein content resembles the parental cells as well as primary tumors. Promising candidates to be transferred by ST-EPN-RELA EVs but not control EVs were associated with unfolded protein response and endoplasmic reticulum stress. When uptaken by recipient cells of the tumor microenvironment, brain endothelial cells or microglia, ST-EPN-RELA EVs induced proliferation and had a chemoattractant effect towards the tumor. ST-EPN-RELA EVs stimulated angiogenesis of brain endothelial cells potentially by the transfer of ER stress proteins. Uptake of ST-EPN-RELA EVs by microglia changed their activation status indicating a tumor promoting function through EV transfer. Therefore, we hypothesize that vesicular pathways play an important role in the pathogenesis of pediatric ST-EPN-RELAs and that an improved understanding may promote new therapeutic opportunities.

EPEN-34. THE CRISPR-Cas9 SYSTEM-MEDIATED ENDOGENOUS GENE REARRANGEMENT INDUCED C11orf95-RELA FUSION IN VITRO AND IN VIVO THAT LED TO THE DEVELOPMENT OF EPENDYMOMA-LIKE TUMOR

Recent large-scale genomic studies of ependymal tumors have identified recurrent RELA and YAP1 fusion genes in supratentorial ependymomas. The formation of the C11orf95-RELA fusion gene has been attributed to massive genomic rearrangement involving chromosome 11q termed Chromothripsis in many cases. However, the causal relationship has not been clarified experimentally. In this study, we developed a system to reproduce the oncogenic gene rearrangement using the CRISPR-Cas9 system and examined whether consequent endogenous ependymoma fusion genes are competent to form brain tumors in mice. Initially, to investigate whether C11orf95-RELA fusion can be formed by inducing the relevant gene rearrangement in vitro, we designed multiple guide RNAs on the human and mouse genomic loci and introduced them into cultured cells. RT-PCR and immunoblot analyses detected endogenous C11orf95-RELA fusion transcript and protein in both human and mouse cultured cells. Subsequently, we lentivirally introduced the gRNAs into a mouse brain. Brain tumor formation was observed from around 2 months after the lentivirus injection, thus indicating successful gene rearrangement followed by C11orf95-RELA fusion expression in vivo. Analysis of the tumor tissue confirmed the expression of the endogenous C11orf95-RELA fusion gene. These results suggested that a gene rearrangement is a primary mechanism to form the C11orf95-RELA fusion which is the direct driver of tumorigenesis. Our system to simulate a genomic event will provide significant insights into the understanding of the tumorigenic mechanism in ependymomas.

EPEN-01. MULTIDISCIPLINARY TREATMENT IN EPENDYMOMA

BACKGROUNDIn intracranial ependymoma, the effectiveness of chemotherapy and radiation therapy is unclear, and the degree of tumor removal contributes to the improvement of life prognosis.METHODSWe examined ependymoma cases treated in our institution from July 1998 to March 2017.RESULTSThere were 18 boys and 7 girls. The average age at the time of surgery is 5.3±3.6 years. The pathological diagnosis was Grade II for 8 cases and Grade III for 17 cases. Genetic analysis was performed in 16/25 cases (64%). Of the infratentorial cases, 10/11 cases (90.1%) were PFA and PFB were one case. Of the supratentorial cases, 3/5 cases (60%) were positive for RELA fusion. As chemotherapy, 19 patients were VCR + VP-16 + CDDP + CPA. Irradiation was performed in all cases, local irradiation (50.4–55.8Gy) in 22 cases (88%), and craniospinal irradiation in 2 cases (8%). The 7-year OS was 74.6±9% and the 7-year PFS was 59.7±10.5%. Grade III showed a short OS (p = 0.053). GTR and NTR were obtained in the first excision in 14 cases (56%), and OS and PFS were not significantly different from those in the STR group (p = 0.219, p = 0.248). GTR and NTR including 2nd-look surgery were obtained in 18 cases (72%), and significant improvement of OS was observed compared with STR group (p = 0.02).CONCLUSIONEven if it is not GTR or NTR at the first operation, improvement of OS is expected by total excision after chemotherapy.

PATH-28. MOLECULAR DIAGNOSIS FOR CENTRAL DIAGNOSIS OF BRAIN TUMORS FROM 2016 TO 2019— A REPORT FROM THE JAPAN CHILDREN’S CANCER GROUP (JCCG)

INTRODUCTIONSince 2016, the Japan Children’s Cancer Group (JCCG) has established a nationwide network that prospectively provides pathological review and molecular analysis.METHODSPatients who were diagnosed with brain tumors between ages 0 and 29 were eligible. The central office at National Center for Child Health and Development served as a hub for the hospitals involved and institutions conducting pathological and molecular analysis, and managed the patients’ clinical information and tumor samples. Histopathology of all cases were centrally reviewed. Routine non-NGS based analyses were conducted based on histological diagnosis and included pyrosequencing for glioma-associated hot spot mutations and PFA/PFB classification for ependymoma, RT-PCR for RELA fusion and BRAF fusion, and nanostring for subgrouping medulloblastoma. In selected cases, methylation analysis, RNA sequencing and exon sequencing of 93 genes were performed in selected cases.RESULTSIn total, 985 cases were registered to this study in four years. Frozen samples were collected from approximately 80% of cases. The number increased from 152 in 2016 to 326 in 2019. They includes glioma (n=268), medulloblastoma (n=161), ependymoma (n=103), germ cell tumor (n=93), ATRT (n=29) and others. In 55 % of the glioma cases, at least one abnormality was detected by the routine analysis. The detailed analysis for atypical cases identified targetable alternations. DISCUSSION: This nationwide central diagnostic system has now been well established. Current issues and future prospective of the system will be discussed.

Phenotypic characterization with somatic genome editing and gene transfer reveals the diverse oncogenicity of ependymoma fusion genes

Recurrent RELA and YAP1 fusions are intimately associated with tumorigenesis in supratentorial ependymomas. Chromothripsis and focal copy number alterations involving 11q are hallmarks of these tumors. However, it is unknown whether the chromosomal alterations are a direct causal event resulting in fusion transcripts. In addition, the biological significance of the RELA fusion variants and YAP1 fusions is not yet fully characterized. In this study, we generated gene rearrangements on 11q with the CRISPR/Cas9 system and investigated the formation of oncogenic ependymoma fusion genes. Further, we examined the oncogenic potential of RELA fusion variants and YAP1 fusions in a lentiviral gene transfer model. We observed that endogenous RELA fusion events were successfully induced by CRISPR/Cas9-mediated genome rearrangement in cultured cells. In vivo genome editing in mouse brain resulted in the development of ependymoma-like brain tumors that harbored the Rela fusion gene. All RELA fusion variants tested, except a variant lacking the Rel homology domain, were able to induce tumor formation, albeit with different efficacy. Furthermore, expression of YAP1-FAM118B and YAP1-MAMLD1 fusions induced the formation of spindle-cell-like tumors at varying efficacy. Our results indicate that chromosomal rearrangements involving the Rela locus are the causal event for the formation of Rela fusion-driven ependymomas in mice. Furthermore, the type of RELA. fusion might affect the aggressiveness of tumors and that the Rel homology domain is essential for the oncogenic functions of RELA. fusions. The YAP1 fusion genes are also oncogenic when expressed in mice.

The Survival and Prognostic Factors of Supratentorial Cortical Ependymomas: A Retrospective Cohort Study and Literature-Based Analysis.

AimSurvival rates and prognostic factors of cortical ependymomas (CEs) remain elusive. This study aimed to perform a comprehensive analysis of prognostic factors, treatment, and outcomes for patients with CEs based on institutional and literature case series.Materials and MethodsThirty patients with CEs from our department were included in this study. Furthermore, a systemic review of the literature yielded an additional 106 patients with CEs. Clinical data including patient age, sex, symptoms, tumor location, World Health Organization (WHO) grade, extent of surgery, radiation, recurrence, and survival were recorded and statistically analyzed.ResultsFrom January 2009 to October 2019, 30 (4.2%) cases were diagnosed as CEs in our department. These series consisted of 19 males and 11 females, 10 continuous patients after 2017 screened for C11orf95-RELA fusion, and 9 patients (90%) were RELA fusion positive. During the follow-up period, nine (30%) patients depicted tumor recurrence or progression; four (13.3%) patients died of tumor progression. The literature review yielded 106 CE cases, with additional 30 cases of our own collected for further analysis. Of these 136 cases, the frontal lobe (40%) was the most common location, and the average age was 22.6 ± 17.6 years. Anaplastic histology/WHO grade III tumors were identified in 68 (50%) patients. Statistically analysis demonstrated that extent of surgery and WHO tumor grade were significant prognostic factors in Kaplan–Meier log-rank testing and Cox proportional hazards models. Gross total resection (GTR) predicted longer progression-free survival (PFS) [P = 0.013, hazard ratio (HR) = 3.012, 95% confidence interval (CI) = 1.257–7.213] and overall survival (OS) (P = 0.003, HR = 5.322, 95% CI = 1.751–16.178). WHO grade III tumors had worse PFS (P = 0.002, HR = 5.17, 95% CI = 1.804–14.816) and OS (P = 0.025, HR = 5.640, 95% CI = 1.248–25.495).ConclusionCEs accounted for only 3.5 to 5.7% of ependymomas, with seizures the most common symptom and the frontal lobe the most frequent location. CEs may have higher rate of RELA fusions, but generally favorable prognosis. The extent of surgery and WHO tumor grade were significant prognostic factors for PFS and OS in multivariate analysis. GTTR or WHO grade II tumors had better overall outcome in patients with CEs.