C11orf95-RELA Fusion Research Articles

ETMR-29. PRIMARY CNS SARCOMAS IN CHILDREN AND ADOLESCENTS: EXPERIENCE OF AN INSTITUTION

Abstract BACKGROUND Soft tissue sarcomas represent 6% of childhood cancers, less than 0.2% of CNS tumors are primary intracranial sarcomas. Their origin is pluripotential primitive mesenchymal cells. OBJECTIVE to evaluate clinical and genetic characteristics and report the experience of a rare CNS tumor. METHODS review of CNS tumors database at IOP/GRAACC/UNIFESP for the period 2008-2024, searching for all types of sarcomas that involved the CNS. RESULTS case series included 7 male, 2 female patients; average age at diagnosis 11.3 years. Supratentorial was the most common site of occurrence (66.6%), posterior fossa and intramedulary (2 and 1 cases, respectively) were found. One PF sarcoma presented as a second neoplasm after 2 years finished treatment for medulloblastoma, the other was diagnosed just after metilation. The presenting symptoms varied with tumor location, specially hemiparesis, seizure and headache. Gross total resection was achieved in 7 cases (77.7%), 1 after second look surgery. Histology and immunohistochemistry studies revealed: spinocellular sarcoma, fusocellular and myxoid sarcoma, gliosarcoma, meningeal sarcoma, Ewing sarcoma, condrosarcoma and synovial sarcoma. Work-up excluded other primary tumor origin outside CNS. Analyses for genetic characteristics were performed by Next Generation Sequencing in 6 patients (66.6%) and the following alterations were found: NRAS, TP53, JAK3, NF2, C11orf95-RELA fusion, PDGFRA. Some of these, by now, with no significant meaning for the disease. All patients underwent multimodal treatment. Chemotherapy involved mainly ICE Protocol (55.5%), but also Head Start Protocol and Ewing Sarcoma Protocol. Follow-up ranged from 13 months to 12 years, 90% of patients are alive, 2 (28.5%) dealing with endocrinal issues after focal radiotherapy. The mean overall survival was 61.4 months. CONCLUSION CNS primary sarcomas were mostly observed in the supratentorial region and in older children, with male predilection. Multimodal treatment seems to benefit patients’ outcomes. More patients and longer follow-up periods are needed to further elucidate the biological features of these tumors.

DDDR-34. RECURRENT RELA FUSION-POSITIVE EPENDYMOMA TREATED WITH VAL-083 UNDER EXPANDED ACCESS: A CASE REPORT

Abstract Ependymoma is a relatively rare central nervous system tumor accounting for 2-9 % of all neuroepithelial tumors. RELA fusion-positive ependymoma is a subgroup associated with supratentorial location, higher WHO grade and worse prognosis. In addition, there is no standard systemic chemotherapy treatment for adults with recurrent disease. VAL-083 is a bi-functional DNA-targeting agent which rapidly induces inter-strand DNA cross-links at N7-guanine inducing double-strand breaks causing cell death and acts independent of MGMT DNA-repair in high-grade gliomas. We report a 40-year-old male who was initially diagnosed with a right parieto-occipital high-grade glioma, with no somatic mutations including IDH1/2 genes, and with unmethylated MGMT promoter status. He initially underwent gross total resection, followed by chemoradiation with concurrent and adjuvant temozolomide for 12 cycles. Sixteen months later, he developed recurrent disease and had a second resection. Inter- and intragenic fusion analysis of tumor tissue revealed C11orf95-RELA fusion and the diagnosis of RELA fusion-positive ependymoma was established. The patient was not eligible to participate in any clinical trial and received VAL-083 under an expanded access program. He was then treated with VAL-083 (30 mg/m2 for 3 consecutive days every 21 days) and completed 12 cycles during a period of 9 months. No grade 3/4 adverse events such as thrombocytopenia, anemia, neutropenia, or lymphopenia were observed. His liver and renal functions were normal. No dose reduction was required. He also received levetiracetam, alprazolam and prochlorperazine, with no drug interactions. Steroid were not required. He has been under surveillance since completion of systemic treatment with VAL-083 and has remained radiologically stable, with no CSF dissemination, after 12 months. This case highlights that VAL-083 may be a treatment option for recurrent RELA fusion-positive ependymoma refractory to temozolomide-based regimens. Clinicaltrials.gov Identifier: NCT03138629. The treatment plans for this EAP patient were approved by MD Anderson Cancer Center IRB.

Clinical and Molecular Features in Medulloblastomas Subtypes in Children in a Cohort in Taiwan.

Medulloblastoma (MB) was classified into four molecular subgroups: WNT, SHH, group 3, and group 4. In 2017, 12 subtypes within 4 subgroups and 8 subtypes within non-WNT/non-SHH subgroups according to the differences of clinical features and biology were announced. In this study, we aimed to identify the heterogeneity of molecular features for discovering subtype specific factors linked to diagnosis and prognosis. We retrieved 70 MBs in children to perform RNA sequencing and a DNA methylation array in Taiwan. Integrated with clinical annotations, we achieved classification of 12 subtypes of pediatric MBs in our cohort series with reference to the other reported series. We analyzed the correlation of cell type enrichment in SHH MBs and found that M2 macrophages were enriched in SHH β, which related to good outcomes of SHH MBs. The high infiltration of M2 macrophages may be an indicator of a favorable prognosis and therapeutic target for SHH MBs. Furthermore, C11orf95-RELA fusion was observed to be associated with recurrence and a poor prognosis. These results will contribute to the establishment of a molecular diagnosis linked to prognostic indicators of relevance and help to promote molecular-based risk stratified treatment for MBs in children.

Supratentorial extra-axial RELA fusion-positive ependymoma misdiagnosed as meningioma by intraoperative histological and cytological examinations: a case report

BackgroundDura-attached supratentorial extra-axial ependymoma is a very rare type of tumor, with only nine reported cases. Preoperative diagnosis of dura-attached supratentorial extra-axial ependymoma is difficult and often radiologically misdiagnosed as a meningioma. We report a case of dura-attached supratentorial extra-axial ependymoma that was misdiagnosed using intraoperative histological and cytological examinations.Case presentationA 26-year-old Japanese man with headache and nausea was referred to our medical facility. Magnetic resonance imaging revealed a cystic mass of 70 × 53 × 57 mm in the left temporoparietal lobe. A peritumoral band with hyperintensity on T2-weighted imaging was observed at the periphery of the lesion, suggesting an extra-axial lesion with no apparent connection to the ventricle. A dural tail sign was also noted on the gadolinium-enhanced T1-weighted image. Preoperative clinical diagnosis was meningioma. Proliferated tumor cells in sheets with intermingled branching vessels were observed in the frozen tissue. Perivascular rosettes were inconspicuous, and the tumor cells had rhabdoid cytoplasm. The tumor was intraoperatively diagnosed as a meningioma, suspected to be a rhabdoid meningioma. Perivascular rosettes were evident in the formalin-fixed paraffin-embedded tissues, suggesting ependymoma. The tumor cells had eosinophilic cytoplasm without a rhabdoid appearance. Anaplastic features, such as high tumor cellularity, increased mitotic activity, microvascular proliferation, and necrosis, were observed. Ependymal differentiation was confirmed on the basis of ultrastructural analysis. Molecular analysis detected C11orf95-RELA fusion gene. The final diagnosis was RELA fusion-positive ependymoma, World Health Organization grade III.ConclusionOwing to its unusual location, dura-attached supratentorial extra-axial ependymomas are frequently misdiagnosed as meningiomas. Neuropathologists should take great precaution in intraoperatively diagnosing this rare subtype of ependymoma to avoid misdiagnosis of the lesion as other common dura-attached 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.

C11orf95-RELA fusion drives aberrant gene expression through the unique epigenetic regulation for ependymoma formation

Recurrent C11orf95-RELA fusions (RELAFUS) are the hallmark of supratentorial ependymomas. The presence of RELA as the fusion partner indicates a close association of aberrant NF-κB activity with tumorigenesis. However, the oncogenic role of the C11orf95 has not been determined. Here, we performed ChIP-seq analyses to explore genomic regions bound by RELAFUS and H3K27ac proteins in human 293 T and mouse ependymoma cells. We then utilized published RNA-Seq data from human and mouse RELAFUS tumors and identified target genes that were directly regulated by RELAFUS in these tumors. Subsequent transcription factor motif analyses of RELAFUS target genes detected a unique GC-rich motif recognized by the C11orf95 moiety, that is present in approximately half of RELAFUS target genes. Luciferase assays confirmed that a promoter carrying this motif is sufficient to drive RELAFUS-dependent gene expression. Further, the RELAFUS target genes were found to be overlapped with Rela target genes primarily via non-canonical NF-κB binding sites. Using a series of truncation and substitution mutants of RELAFUS, we also show that the activation domain in the RELAFUS moiety is necessary for the regulation of gene expression of these RELAFUS target genes. Lastly, we performed an anti-cancer drug screening with mouse ependymoma cells and identified potential anti-ependymoma drugs that are related to the oncogenic mechanism of RELAFUS. These findings suggested that RELAFUS might induce ependymoma formation through oncogenic pathways orchestrated by both C11orf95 and RELA target genes. Thus, our study unveils a complex gene function of RELAFUS as an oncogenic transcription factor in RELAFUS positive ependymomas.

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-53. C11orf95-RELA REPROGRAMS 3D EPIGENOME IN SUPRATENTORIAL EPENDYMOMA

Ependymoma is the third most common malignant brain tumor in children. However, there is no effective chemotherapy identified and treatment is limited to surgery with or without adjuvant radiation therapy currently. Thus, to develop targeted therapy based on the underlying biology is an urgent need. Since 2014, C11orf95-RELA fusion was found to be the most recurrent structural variation in approximately 70% of supratentorial ependymomas (ST-EPN), but the molecular mechanisms of oncogenesis are unclear. Here we utilized HEK293T transgene models and a ST-EPN cell line to investigate the epigenomic changes and transcriptional regulations by C11orf95-RELA fusion. By applying ChIP-seq and HiChIP approaches, we found C11orf95-RELA is a novel transcription factor that recognizes a specific DNA motif dictated by the C11orf95 component while the RELA component is required for driving the expression of ependymoma-associated genes such as CCND1 and L1CAM. Moreover, C11orf95-RELA modulates chromatin states and mediates chromatin interactions, leading to transcriptional reprogramming in ST-EPN cells. Multiple signaling pathways such as Notch signaling and G-protein signaling are identified to be involved in ST-EPN development. Our findings provide important characterization of the molecular underpinning of C11orf95-RELA fusion and shed light on potential therapeutic targets for C11orf95-RELA subtype ependymoma.

EPEN-30. C11ORF95-RELA FUSION PROTEIN ENGAGES NOVEL GENOMIC LOCI TO DRIVE MURINE EPENDYMOMA GROWTH

RATIONALEOver 70% of supratentorial (ST) ependymoma are characterized by an oncogenic fusion between C11ORF95 and RELA. C11ORF95-RELA fusion is frequently the sole genetic driver detected in ST ependymoma, thus ranking this genomic event as a lead target for therapeutic investigation. RELA is a transcription factor (TF) central to mediating NF-kB pathway activation in processes such as inflammation, cellular metabolism, and chemotaxis. HYPOTHESIS: We posited that C11ORF95-RELA acts as an oncogenic TF that aberrantly shapes the tumor epigenome to drive aberrant transcription. Approach: To this end we developed an in utero electroporation (IUE) mouse model of ependymoma to express C11ORF95-RELA during embryonic development. Our IUE approach allowed us to develop C11ORF95-RELA driven tumor models and cell lines. We comprehensively characterized the epigenome and transcriptome of C11ORF95-RELA fusion driven mouse cells by H3K27ac ChIP-seq, ATAC-seq, and RNA-seq.RESULTSThis data revealed that: 1) C11ORF95-RELA directly engages ‘open’ chromatin and is enriched at regions with known RELA TF binding sites as well as novel genomic loci/motifs, 2) C11ORF95-RELA preferentially binds to both H3K27ac (active) enhancers and promoters, and 3) Bound C11ORF95-RELA promoter loci are associated with increased transcription of genes shared with human ependymoma.CONCLUSIONOur findings shed light on the transcriptional mechanisms of C11ORF95-RELA, and reveal downstream targets that may represent cancer dependency genes and molecular targets.

EPEN-36. THE TREATMENT OUTCOME OF PAEDIATRIC SUPRATENTORIAL C11ORF95-RELA FUSED EPENDYMOMA: A COMBINED REPORT FROM E-HIT SERIES AND AUSTRALIAN NEW ZEALAND CHILDREN’S HAEMATOLOGY/ONCOLOGY GROUP

AIMAdvances in molecular classification of paediatric ependymoma have been pivotal in improving risk stratification and understanding of this disease. C11orf95-RELA fused supratentorial ependymoma (ST-EPN) have been reported to have a poor outcome, with 10-year overall survival (OS) of 49% and progression free survival (PFS) of 19%. A cohort of patients from multiple international institutions with molecularly confirmed C11orf95-RELA fused ST-EPN were reviewed to assess their disease behaviour. METHOD: We reviewed patients with molecularly determined C11orf95-RELA supratentorial ependymoma diagnosed between 1999 – 2019. Demographic information, extent of surgical resection, use of radiotherapy and/or chemotherapy, disease recurrence, treatment at recurrence and clinical outcome data was collected. PFS and OS of all patients were estimated using Kaplan-Meier method.RESULTSA total of 76 ST-EPN patients with C11orf95-RELA fusion were identified (median age: 7 years3 months, range: 5 months – 18 years7 months). 58 patients (76.3%) had complete surgical resection. 70 patients(92.1%) received radiotherapy. 55 patients(72.3%) received chemotherapy. The 10-year OS of C11orf95-RELA fused ST-EPN was 72.4% and PFS was 63.8%. In contrast, ST-EPN at a single institution with unconfirmed molecular status had an OS of 61.1% and PFS of 34.9%.CONCLUSIONDetailed molecular analysis identified distinct subgroups of patients with ST-EPN. Patients from this cohort with C11orf95-RELA methylation profiles had a significantly higher OS compared to previous reports and those with unconfirmed fusion status, emphasising the critical importance of complete molecular profiling to assist in treatment decision making. Complete molecular analysis in future prospective cohorts is essential for accurate risk stratification and treatment selection.

EPEN-41. C11orf95-RELA FUSION REGULATES ABERRANT GENE EXPRESSION THROUGH THE UNIQUE GENOMIC BINDING SITES FOR EPENDYMOMA FORMATION

A majority of supratentorial ependymoma is associated with recurrent C11orf95-RELA fusion (RELAFUS). The presence of RELA as one component of the RELAFUS leads to the suggestion that NF-kB activity is involved in the ependymoma formation, thus being a viable therapeutic target in these tumors. However, the oncogenic role of another C11orf95 component in the tumorigenesis is not still determined. In this study, to clarify the molecular mechanism underlying tumorigenesis of RELAFUS, we performed RELAFUS-ChIP-Seq analysis in cultured cells expressing the RELAFUS protein. Genomic profiling of RELAFUS binding sites pinpointed the transcriptional target genes directly regulated by RELAFUS. We then identified a unique DNA binding motif of the RELAFUS different from the canonical NF-kB motif in de novo motif discovery analysis. Significant responsiveness of RELAFUS but not RELA to the motif was confirmed in the reporter assay. An N-terminal portion of C11orf95 was sufficient to localize in the nucleus and recognizes the unique motif. Interestingly, the RELAFUS peaks concomitant with the unique motif were identified around the transcription start site in the RELAFUS target genes as previously reported. These observations suggested that C11orf95 might have served as a key determinant for the DNA binding sites of RELAFUS, thereby induced aberrant gene expression necessary for ependymoma formation. Our results will give insights into the development of new ependymoma therapy.

C11orf95-RELA reprograms 3D epigenome in supratentorial ependymoma

Supratentorial ependymoma (ST-EPN) is a type of malignant brain tumor mainly seen in children. Since 2014, it has been known that an intrachromosomal fusion C11orf95-RELA is an oncogenic driver in ST-EPN [Parker et al. Nature 506:451–455 (2014); Pietsch et al. Acta Neuropathol 127:609–611 (2014)] but the molecular mechanisms of oncogenesis are unclear. Here we show that the C11orf95 component of the fusion protein dictates DNA binding activity while the RELA component is required for driving the expression of ependymoma-associated genes. Epigenomic characterizations using ChIP-seq and HiChIP approaches reveal that C11orf95-RELA modulates chromatin states and mediates chromatin interactions, leading to transcriptional reprogramming in ependymoma cells. Our findings provide important characterization of the molecular underpinning of C11orf95-RELA fusion and shed light on potential therapeutic targets for C11orf95-RELA subtype ependymoma.

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.

Abstract B71: Molecular heterogeneity and novel oncogenic fusions in RELA- and YAP1-negative supratentorial ependymoma

Abstract Introduction: One of the DNA methylation-based molecular subgroups of supratentorial ependymoma (ST-EPN), designated ST-EPN-RELA, mostly harbors fusions of the uncharacterized gene C11orf95 and RELA (ST-EPN-RELA). Rarely, no C11orf95-RELA fusion is detected in tumors predicted to belong to the ST-EPN-RELA group. With this study we aimed to refine the molecular classification of ST-EPN and to identify alternative oncogenic mechanisms in the absence of a classic fusion type. Methods and Materials: In an unbiased approach, t-Distributed Stochastic Neighbor Embedding was applied to 53,468 DNA methylation profiles from brain tumors, other cancer types, and control tissues. Only samples clustering with a reference set of ST-EPN-RELA were selected for further analyses (n=614), including RNA- and/or DNA-panel sequencing, histopathologic reevaluation, and immunohistochemistry for L1CAM. Fusions were validated using RT-PCR on total RNA and Sanger sequencing. Clinical data were analyzed retrospectively for 150 patients. Results: We identified one large and four satellite clusters. The large cluster (n=479; designated ST-EPN-RELA 1) and one of the satellite clusters (n=12; ST-EPN-RELA 2) predominantly contained samples with a calibrated score ≥ 0.9 for ST-EPN-RELA based on the current version of the Heidelberg Brain Tumor Classifier. Samples of the three other satellite clusters (n=41, n=17, and n=25 samples) contained 65.9%, 88.2%, and 96.0% of samples with a calibrated score < 0.9 for any methylation class, and were thus predicted as unclassifiable. These clusters were provisionally designated ST-EPN-RELA-like A, B, and C, and initial histologic diagnoses showed a wide spectrum of rare morphologies beside EPN, e.g., sarcoma and teratoma. Within clusters ST-EPN-RELA-like A and C, sequencing revealed fusions of C11orf95 with different partner genes, including MAML2 (n=14), MAML3 (n=2), and NCOA2 (n=7), while ST-EPN-RELA-like B included classic C11orf95-RELA fusions (n=11) in samples with initial diagnoses other than EPN. Copy number variation analysis showed clear differences between the clusters. L1CAM-positivity was observed in all groups. Within the cluster ST-EPN-RELA 1, samples separated according to fusion types, 1 versus 2/3. Analysis of clinical data showed significant differences in overall survival between cases with confirmed C11orf95-RELA fusion type 1 (n=25, median OS=88 months) and type 2/3 (n=20, median OS=67 months). Clinical data collection for the satellite clusters is currently ongoing. Conclusion: Molecular refinement of ST-EPN-RELA revealed novel subgroups harboring fusions of C11orf95 with numerous fusion partners different from RELA, which will be included in the next update of the Heidelberg Classifier. Preliminary analysis suggests differences in clinical outcome related to the fusion type. Findings of this study will improve diagnostic accuracy and clinical management and need to be considered when developing targeted treatment strategies against ST-EPN. Citation Format: D.R. Ghasemi, K. Okonechnikov, A. Korshunov, M. Sill, T. Zheng, J.M. Huebner, K.K. Maass, J. Benzel, M. Snuderl, J. Gojo, U. Schüller, N.U. Gerber, I. Stoler, P. Hernáiz-Driever, T. Milde, D. Sturm, R. Chapman, R.G. Grundy, A. von Deimling, D. Kawauchi, D.T.W. Jones, M. Kool, S.M. Pfister, F. Sahm, K.W. Pajtler. Molecular heterogeneity and novel oncogenic fusions in RELA- and YAP1-negative supratentorial ependymoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B71.

Supratentorial pediatric cortical ependymomas: a comprehensive retrospective study.

Pediatric cortical ependymomas (CEs) are rare; the clinical features and optimal treatment remain ill-defined. We aimed to clarify the clinical characteristics and outcome of pediatric CEs based on institutional series and literature review. Thirteen children with CEs from our department were included in the present study. Furthermore, a search of English language peer-reviewed articles yielded 43 patients with CEs. The clinical data, treatment, and outcome were retrospectively reviewed and statistically analyzed. Our institutional series consisted of nine males and four females. The literature review yielded 56 pediatric CE cases (including ours) for further analysis. Of these 56 cases, frontal lobe (n = 19, 41.3%) was the most common location and most of the tumors were located in the right hemisphere (n = 27, 58.7%). Seizures (n = 23, 41.1%) were the most frequent preoperative symptoms. Thirty patients (n = 30, 53.6%) were WHO grade II. Five continuous patients in our series screened for C11orf95-RELA fusion and all the patients (100%) were RELA fusion positive. Fourteen (26.4%) patients experienced tumor recurrence and 4 (7.5%) patients died during the follow-up. Multivariate survival analysis depicted extent of surgery resection was the only prognostic factor for PFS and patient with gross total resection (P = 0.037, HR 3.682, 95% CI 1.082-13.79) had longer PFS. Furthermore, Log-rank testing for Kaplan-Meier survival analysis showed the extent of surgery resection (P = 0.007) was the only prognostic factor for OS. Pediatric CEs are rare, commonly seen in frontal lobe and right hemisphere. Seizures are the most common symptoms. They may have higher rate of RELA fusions, but favorable outcome. A low incidence of anaplastic histology has been depicted. Gross total resection is significantly associated with longer PFS and OS. Careful follow-up is necessary because the tumors may progress.

L1CAM High Expression Associates with Poor Prognosis in Glioma but Does Not Correlate with C11orf95-RELA Fusion.

The latest WHO guideline of CNS tumor defined a RELA fusion-positive ependymoma type with extremely poor prognosis, and the expression of L1CAM was correlated well with the presence of RELA fusion. However, the L1CAM protein expression in large sample gliomas other than ependymoma, its relationship with the RELA gene and its prognostic significance remained unknown. We examined the expression of L1CAM in 565 glioma cases (WHO grade I-IV). The L1CAM IHC-positive cases were selected to test RELA fusion with FISH break-apart probes. L1CAM was positive in 109 cases (19.29%) of all 565 glioma cases, with 18.27% in low-grade gliomas and 19.84% in high-grade gliomas, respectively. Unlike ependymoma, L1CAM protein expression was not correlated with the C11orf95-RELA fusion gene in other gliomas, but it had correction with the patient age (older than 45-year-old, p = 0.006), ATRX mutation (p = 0.003) and Ki67 (p = 0.007). High expression of L1CAM was an independent prognostic factor in our cohort. Further analysis demonstrated that L1CAM strong positive expression was significantly associated with poor prognosis in gliomas, both in our cohort (p < 0.001) and TCGA (p < 0.009) dataset. Although uncorrelated with C11orf95-RELA fusion, L1CAM was a significant poor prognostic marker in glioma patients. More aggressive treatment should be taken for these patients and L1CAM might be a promising therapeutic target in glioma.

Multiplatform Molecular Profiling Reveals Epigenomic Intratumor Heterogeneity in Ependymoma

SUMMARYEpendymomas exist within distinct genetic subgroups, but the molecular diversity within individual ependymomas is unknown. We perform multiplatform molecular profiling of 6 spatially distinct samples from an ependymoma with C11orf95-RELA fusion. DNA methylation and RNA sequencing distinguish clusters of samples according to neuronal development gene expression programs that could also be delineated by differences in magnetic resonance blood perfusion. Exome sequencing and phylogenetic analysis reveal epigenomic intratumor heterogeneity and suggest that chromosomal structural alterations may precede accumulation of single-nucleotide variants during ependymoma tumorigenesis. In sum, these findings shed light on the oncogenesis and intratumor heterogeneity of ependymoma.

PDTM-27. THE CRISPR-Cas9 SYSTEM-MEDIATED ENDOGENOUS GENE-REARRANGEMENT INDUCED C11orf95-RELA FUSION IN VITRO AND IN VIVO THAT LED TO DEVELOPMENT OF BRAIN TUMORS

Abstract Recent large-scale genomic studies of ependymal tumors have identified recurrent RELA and YAP1 fusion genes in supratentorial ependymomas. We have so far shown that C11orf95-RELA fusion was a potent oncogene capable of inducing human ependymoma-like tumors in the RCAS/tv-a retroviral gene transfer system. However, molecular mechanism underlying the tumor formation has not been fully investigated. Given that C11orf95-RELA fusion genes are caused by gene rearrangement between C11orf95 and RELA gene due to chromothripsis involving 11q, it has been suggested that expression of these fusion genes is regulated under the C11orf95 promoter. In this study, we developed a system to reproduce the oncogenic gene rearrangement using the CRISPR-Cas9 system and examined if consequent endogenous ependymoma fusion genes are competent to form brain tumor in mice. Initially, we designed guide RNAs on the human and mouse genomic loci corresponding to breakpoints observed in human RELA fusion positive ependymomas and introduced them into HEK293T or NIH3T3 cells. RT-PCR and immunoblot analyses revealed that a combination of multiple guide RNAs efficiently induced gene rearrangement between C11orf95 and RELA gene, resulting in C11orf95-RELA fusion protein. Subsequently, we introduced the same guide RNAs using a lentiviral gene transfer system into mouse brain. Brain tumor formation was observed around 2 months after the guide RNA delivery, indicating successful gene rearrangement followed by C11orf95-RELA fusion expression in vivo. These results thus firmly establish that a gene rearrangement is the mechanism to form the C11orf95-RELA fusion which is the direct driver of tumorigenesis. Furthermore, we found that endogenous YAP1-FAM118B fusion gene can be also induced with a similar experimental strategy to that of C11orf95-RELA fusion. Our system to simulate genomic event will provide insights into the tumorigenic mechanism of ependymomas that harbor various fusion genes.

PDTM-29. STRUCTURE FUNCTION AND CELL TYPE DETERMINANTS OF C11orf95-RELA DRIVEN TUMORS IN MICE

Abstract We used a new mouse model to better understand the cellular and molecular determinants of tumors driven by the C11orf95-RELA fusion. Our approach makes use of in utero electroporation and a binary transposase system to introduce human C11orf95-RELA sequence, wild type and mutant, into neural progenitors, and drive expression of the fusion in different glial and neuronal progenitor cell types. Our results indicate that truncations or point mutations in C11orf95 sequence which interfere with nuclear localization result in a complete loss of tumor-inducing activity. The mutations include truncations of the first 60 amino acids, internal truncations that delete possible mono and bipartite nuclear localization signals, and point mutations of two cysteines and histidines that make up a possible zinc finger domain in C11orf95. Interestingly, all of the mutations that block tumorigenesis also block signal independent nuclear localization of the wild type fusion, without blocking induction of NFKB response genes. We further found that over-expression of the NFKB1 subunit P50 which lacks a transcriptional activation domain significantly inhibits tumor formation by the fusion. In addition, we find that driving expression of the wild type fusion in glial progenitor types using promoters for either astrocytes or oligodendrocytes results in the formation of tumors with transcriptomes displaying significant similarities to human supratentorial ependymoma (ST-EPN), but with distinct patterns depending upon the glial progenitor promoter utilized. In contrast, promoters driving expression selectively in neuron restricted progenitors do not result in the formation of ST-EPN. Together our results reveal three new features of C11orf95-RELA driven tumorigenesis: i) multiple sequences within the C11orf95 domain are required for oncogenic driver activity of the fusion, ii) the P50 subunit of NFKB1 can inhibit fusion induced tumorigenesis, and iii) neuron-restricted precursors are less competent than glia-restricted precursors to form tumors induced by C11orf95-RELA.

Pediatric Supratentorial Ependymoma: Surgical, Clinical, and Molecular Analysis.

Pediatric supratentorial ependymomas (SEs) have distinct molecular and behavioral differences from their infratentorial counterparts. To present our experience with pediatric SEs over a 24-yr period. Clinical, operative, and radiographic information was abstracted retrospectively. Our primary outcomes were progression-free survival (PFS) and overall survival (OS). Detection of C11orf95-RELA rearrangement was performed using interphase fluorescence in situ hybridization (iFISH). Seventy-three patients were identified (41 female, 32 male); median age was 6.7 yrs (range, 1 mo-18.8 yr); median follow-up was 8.3 yrs (range, 2.0-26.3). Fifty-eight (79.5%) of 73 patients underwent gross total resection (GTR); no patient with subtotal resection had greater than 1 cm3 of residual tumor; 42 patients (57.5%) experienced subsequent disease progression with 17 patients ultimately dying of their disease. Median PFS was 3.7 yrs. Molecular analysis was available for 51 patients (70%). On bivariate analysis, PFS and OS were not statistically affected by age, tumor grade, or extent of resection, although there was a clinically significant trend for the latter in favor of aggressive resection on PFS (P=.061). Children with RELA fusion had significantly higher PFS (P=.013) than those without, although there was no difference in OS when compared with those with no C11orf95-RELA fusion or C11orf95 gene rearrangement alone. In our series, GTR may be associated with better PFS, but did not impact OS. Surprisingly, RELA fusion was not found to be a negative prognostic factor, raising the possibility that the deleterious effects may be overcome by aggressive resection.