Supratentorial Ependymoma Research Articles

A multi-institutional retrospective pooled outcome analysis of molecularly annotated pediatric supratentorial ZFTA-fused ependymoma.

ZFTA-RELA (formerly known as c11orf-RELA) fused supratentorial ependymoma (ZFTAfus ST-EPN) has been recognized as a novel entity in the 2016 WHO classification of CNS tumors and further defined in the recent 2021 edition. ZFTAfus ST-EPN was reported to portend poorer prognosis when compared to its counterpart, YAP1 ST-EPN in some previously published series. The aim of this study was to determine the treatment outcome of molecularly confirmed and conventionally treated ZFTAfus ST-EPN patients treated in multiple institutions. We conducted a retrospective analysis of all pediatric patients with molecularly confirmed ZFTAfus ST-EPN patients treated in multiple institutions in 5 different countries (Australia, Canada, Germany, Switzerland, and Czechia). Survival outcomes were analyzed and correlated with clinical characteristics and treatment approaches. A total of 108 patients were collated from multiple institutions in 5 different countries across three continents. We found across the entire cohort that the 5- and 10-year PFS were 65% and 63%, respectively. The 5- and 10-year OS of this cohort of patients were 87% and 73%. The rates of gross total resection (GTR) were high with 84 out of 108 (77.8%) patients achieving GTR. The vast majority of patients also received post-operative radiotherapy, 98 out of 108 (90.7%). Chemotherapy did not appear to provide any survival benefit in our patient cohort. This is the largest study to date of contemporaneously treated molecularly confirmed ZFTAfus ST-EPN patients which identified markedly improved survival outcomes compared to previously published series. This study also re-emphasizes the importance of maximal surgical resection in achieving optimal outcomes in pediatric patients with supratentorial ependymoma.

Ependymomas

Ependymal neoplasms are a heterogenous group of neoplasms arising from the progenitors of the cells lining the ventricular system and the spinal central canal. During the last few years, significant novel data concerning oncogenesis, molecular characteristics and clinical correlations of these tumours have been collected, with a strong relevance for their pathological classification. The recently published 5th edition of WHO Classification of Central Nervous System Tumours integrates this novel knowledge and represents a substantial update compared to the previous edition. Concerning supratentorial ependymomas, the previous RELA fusion-positive ependymoma has been renamed into ZFTA fusion-positive and the novel YAP1 fusion-positive ependymoma subtype has been added. Posterior fossa ependymomas should now be allocated either to the Type A or Type B subtypes based on molecular profiling or using the H3 K27me3 immunohistochemical surrogate. Regarding spinal ependymomas, a novel subtype has been added based on a distinctive molecular trait, presence of MYCN amplification, and on the unfavourable outcome. Finally, myxopapillary ependymoma is now classified as a grade 2 tumour in accordance with its overall prognosis which mirrors that of conventional spinal ependymomas. The aim of this review is to present these changes and summarize the current diagnostic framework of ependymal tumours, according to the most recent updates.

Patterns of Extraneural Metastases in Children With Ependymoma.

Ependymomas account for 10% of all malignant pediatric central nervous system tumors. Standard therapy includes maximal safe surgical resection, followed by focal radiation. Despite the aggressive therapy, progression-free survival is poor. Most ependymoma relapses occur locally at the original tumor site. Extraneural presentations of ependymoma are extremely rare, and no standard of care treatment exists. We present a single-institution case series of 3 patients who experienced extraneural relapses of supratentorial ependymoma and describe their treatment and outcome. These cases of extraneural relapse highlight the possible modes of extraneural spread, including hematogenous, lymphatic, and microscopic seeding through surgical drains and shunts. In addition, they illustrate the increase in histologic grade and mutational burden that may occur at the time of relapse. These cases illustrate the role of aggressive, individualized treatment interventions using a combination of surgery, radiation, and chemotherapy.

Extraventricular site indicates higher grade but better prognosis in adult supratentorial ependymomas: a 14-year single-center retrospective cohort.

Supratentorial extraventricular ependymoma (STEE) and supratentorial intraventricular ependymoma (STIE) are two subsets of supratentorial ependymoma (SE). These two subsets have similar gene features and only differ in original sites: STEE occurs in the brain parenchyma, and STIE is located in ventricles and surrounded by cerebral spinal fluid. The present study aims to depict the diversities of these two subsets and elucidate the potential effects of the anatomic site on the tumor with the same type, grade, and molecular features. Sixty-six consecutive adult SE patients from 2008 to 2021 were enrolled in our study. Clinical data, pathological features, and long-term outcomes were analyzed retrospectively. Results demonstrated that adult STEE presented with a higher proportion of WHO grade 3 (P = .028) and higher Ki-67 index (≥10%) (P = .019) compared to adult STIE. Survival analysis demonstrated that patients of grade 3 STEE exhibited a significantly longer overall survival (OS) than patients of grade 3 STIE (median OS, 24.4 months vs. 13.0 months; P = .004). Grade 2 (hazard ratio (HR) = 0.217; P < .001) and gross total resection (GTR) (HR = 0.156; P < .001) were identified as favorable prognostic factors for all adult SE. The STEE was also associated with a lesser hazard of death for patients of grade 3 on multivariate analysis (HR = 0.263; P = .047). These findings suggested that the extraventricular site was an indicator for higher grade and better prognosis in adult supratentorial ependymoma.

JS06.4.A Intracranial ependymomas of the adult: outcome and response to treatments across molecular subtypes

Abstract Background The 2021 WHO Classification lists two molecularly defined types of supratentorial ependymomas (STEs), i.e., ZFTA and YAP1 fusions, and posterior fossa ependymomas (PFEs), i.e, PFA and PFB. Based on retrospective data, the presence of the ZFTA fusion (for STEs) and the PFA subtype (for PFEs) seem to correlate with a poorer outcome. However, prospective data on large cohorts of adult patients are lacking, and the role of treatments is uncertain. The aim of our study is to investigate the clinical characteristics, response to treatment, and outcome of a cohort of adult patients with supratentorial and posterior fossa ependymomas across different molecular subtypes. Patients and Methods Clinical data of patients ≥18 years with STEs and PFEs were retrospectively collected from 2 Italian Centres (Turin, Treviso). ZFTA and YAP1 fusions were detected by FISH, while PFA and PFB subtypes were defined by anti-H3K27me3 immunohistochemistry. Results We collected 42 adult patients with STEs (11, 26.2%) and PFEs (31, 73.8%) diagnosed between 1984 and 2021. Median age was 45 years. ZFTA and YAP1 fusions were found in 5 (45.5%) and 1 (9.1%) case of STEs. PFA and PFB subtypes accounted for 9 (29.0%) and 22 (71.0%) cases of PFEs. Extent of resection (EOR) was gross-total (GTR) in 6/11 (54.8%) STEs and 17/31 PFEs (54.8%). 4/11 (36.4%) STEs and 9/31 (29.0%) PFEs received adjuvant radiotherapy (RT). Median progression-free survival (mPFS) and overall survival (mOS) were 172 and 61.6 months for STEs patients, and not reached (NR) and 332 months for PFEs. For patients with STEs, the presence of ZFTA fusion correlated with a significant shorter PFS (64.0 months vs NR, p = 0.05) and with a trend for shorter mOS (168.0 months vs NR, p = 0.307). The only patient with YAP1 fusion had a very long PFS (33 years). In a multivariable analysis, EOR and adjuvant RT did not significantly affect survival of STEs patients.For patients with PFEs, PFA and PFB subtypes did not differ significantly in terms of mPFS (NR vs 137.0 months, p = 0.513) and mOS (NR vs NR, p = 0.132). Conversely, GTR was associated with a significantly longer mPFS (NR vs 63.0 months, p = 0.007) and with a trend for longer mOS (NR vs 332.0 months, p = 0.146). In a multivariable analysis, GTR was associated with a significantly lower risk of disease progression, both in the entire cohort of PFE patients (p = 0.016), and within the PFA subtype (p = 0.013). Similarly, GTR was associated with a trend for better PFS within the PFB subtype. Conclusion Our preliminary data on a real-life cohort of adult patients confirm the worse prognosis of STEs harbouring the ZFTA fusion and suggest an impact of the EOR among PFEs regardless of molecular subtypes. Larger populations of patients are needed to better define the role of treatment modalities within molecular subrogups. The study is still ongoing in a multicentric setting.

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.

Driver mutations in ADGRL3 are involved in the evolution of ependymoma

Although there have been recent advances in the molecular pathology of ependymomas, little is known about the underlying molecular evolution during its development. Here, we assessed the clinical, pathological and molecular evolutionary process of ependymoma recurrence in a 9-year-old patient who had seven recurrences of supratentorial ependymoma and died from intracranial multiregional recurrences at the age of 19 years old. Whole-genome sequencing (WGS) of 7 tumor samples (1 primary and 6 subsequent recurrent tumors) was performed to elucidate the mutation landscape and identify potential driver mutations for tumor evolution. The genetic profiles of the seven tumor specimens showed significant heterogeneity and suggested a highly branched evolutionary pattern. The mutational signatures and chromothripsis changed with treatments. Strikingly, adhesion G protein-coupled receptor L3 (ADGRL3, also known as Latrophilins 3, LPNH3) was found to be consistently mutated during the entire disease process. However, Sanger sequencing of other 78 ependymoma patients who underwent surgery at our institution showed no genetic alteration of ADGRL3, as found in the present case. The mRNA levels of ADGRL3 were significantly lower in ependymomas (n = 36), as compared with normal brain tissue (n = 3). Grade III ependymomas had the lowest ADGRL3 expression. Moreover, ependymomas with lower mRNA level of ADGRL3 had shorter overall survival. Our findings, therefore, demonstrate a rare evolutionary process of ependymoma involving ADGRL3.

A rare case of the supratentorial extraventricular anaplastic ependymoma. Case report and literature review

Ependymoma is a brain tumor accounting for 1.9 % of all benign brain tumors and 3.1 % of glial tumors and 2–9 % of all neuroepithelial tumors. Approximately one third of intracranial ependymal tumors are supratentorial. They may be attached to the ependymal walls of the III ventricle and lateral ventricles or may be sited in the white matter without direct connection to the ventricular system pressing the adjacent cortex. In very rare cases ependymomas may lie cortically with blood supply from the dura – so-called «cortical» ependymomas.Posterior fossa ependymomas are more common in pediatric population with mean age of 6 years, whereas supratentorial ependymomas manifest in adults. In adults Grade III anaplastic ependymomas are most commonly seen.These tumors have no specific features and clinical manifestations of extraventricular anaplastic ependymomas may vary greatly depending on localization and size of the tumor.Supratentorial anaplastic ependymomas have no specific neuroimaging features either. Tumors are often hypo- and isointense in T1 and iso- or hyperintense in T2 sequence, Gd -inhancement is variable.Ependymomas are considered to be non-invasive and to have a strict border with brain tissue thus leading to clinical manifestations because of mass-effect.Surgical resection is the main treatment option for ependymoma. Patients with local Grade II tumor recurrence and patients with Grade III ependymomas should under go radiotherapy on the tumor r esection cavity.We present a rare case of a supratentorial extraventricular anaplastic ependymoma. A 21‑year-old female presented to the clinic with paresthesia and numbness in right hand, right half of lower lip and astereognosis. The magnetic resonance imaging of the brain showed a mass lesion in left fronto-parietal region. At 10.10.2017 surgery was performed and tumor was resected. Histological findings matched with features of anaplastic ependimoma, WHO Grade III. There was no tumor progression during 3 months after surgery.

INSP-02. WHO 2021 classification of CNS tumors

In line with recommendations of the cIMPACT-NOW consortium, the fifth edition of the WHO Classification of Tumors of the Central Nervous System (WHO CNS5 classification) is substantially different from the previous (revised 4th) edition. Salient changes include the separation of pediatric-type low- and high-grade diffuse gliomas from adult-type diffuse gliomas, refinement of the classification of ependymal tumors, and the addition of a newly recognized embryonal CNS tumors. Furthermore, for some tumors the name was changed. For example, diffuse midline glioma (DMG), H3K27M-mutant is now DMG, H3K27-altered (because there are H3-wildtype DMGs that do show loss of nuclear H3K27me3 staining and with a similar prognosis as DMGs, H3K27M-mutant), and supratentorial ependymoma, RELA fusion-positive was changed into ZFTA fusion-positive (as ZFTA (‘zinc finger translocation associated’, the new name for c11orf95) is the more frequent fusion partner in these tumors). The WHO CNS5 tumor classification certainly is an improvement, but it brings several (new) challenges as well. For example, for more CNS tumors it is now impossible to reach a state-of-the-art ‘histomolecular’ diagnosis in case molecular tools for assessment of essential diagnostic characteristics are not available. In those situations, adding NOS (not otherwise specified) to the histology-based diagnosis is the way to go. Furthermore, designing the optimal therapeutic management for newly defined tumor types is challenging. And while a more precise classification facilitates enrollment of more homogeneous populations of patients in clinical studies, the higher granularity of CNS tumor taxonomy makes it more difficult to perform studies on a large number of patients for particular tumor types. Still, one would like to think that patients suffering from a CNS tumor are better served by a more precise diagnosis because this allows for a better estimation of prognosis and, hopefully sooner than later, for a more tailored and effective therapeutic approach.

EPEN-09. Multi-omics characterization of the blood-brain barrier in molecular groups of ependymoma

Abstract BACKGROUND: A major challenge in the treatment of brain tumors is the limited penetration of many drugs through the blood-brain barrier (BBB). BBB characteristics include endothelial cells connected by tight junction proteins (e.g. Claudin5) and expression of efflux transporters (e.g.P-gp) with the physiological role to limit the accumulation of potentially toxic substances in brain tissue. While BBB permeability shows anatomical region-specific characteristics and changes with age, brain tumors can also impact its integrity. To achieve therapeutically relevant drug concentrations in the tumor, characterization of the pathophysiological BBB is fundamental when developing (pre)clinical trials. Our study seeks to increase our understanding of the BBB composition in various molecular groups of ependymoma. RESULTS: Transcriptional BBB characteristics were assessed for primary ependymoma (n=440), cell lines (n=3), mouse models (n=22), and healthy brain controls (n=200). T-distributed stochastic neighbor embedding (tSNE)-based clustering analyses based on most relevant tight junction and efflux transporter gene sets revealed distinct molecular ependymoma group-specific expression patterns. While patient-derived xenografts models (PDX, n=20) showed high similarity with patient tumor samples, in utero electroporation-based (IUE, n=2) mouse models did not fully recapitulate these BBB characteristics. Supratentorial ependymoma with ZFTA fusions revealed a higher transcriptional expression level of important tight junctions (e.g.Claudin5) compared to other groups and normal brain which was confirmed at protein level in respective PDX models by using both western blot and ultra-high content imaging. CONCLUSION: Analyses of important BBB markers revealed significant differences between molecular groups of ependymoma, which may partly explain drug resistance of ependymoma with ZFTA fusion caused by a low BBB permeability. BBB characteristics of corresponding models suggest that IUE and PDX models representing ZFTA fusion-driven tumors cannot equally be used within preclinical drug trials. Findings will be further validated in preclinical studies while molecular BBB characterization will be expanded to other brain tumors.

EPEN-20. PDL1 expression and tumor-infiltrating immune cell subpopulations: The role of immune-checkpoint molecules in the prognosis of patients with supratentorial ependymoma and its correlation to survival

Immune checkpoint blockade of the programmed cell death ligand-1 (PD-L1)/programmed death-1 (PD-1) axis has shifted the way advanced cancers are treated. The 'ST-RELA fusion positive' and 'PF-A' molecular subgroups of ependymomas (EPN) have a dismal prognosis. AIMS: This study is undertaken to look for immunological microenvironment in ependymomas. Based on L1CAM protein expression and/or the presence of RELA/YAP1 fusion transcripts by RT-PCR, all supratentorial (ST) Grade-II/III EPNs were classified as ST-RELA, ST-YAP, and ST-not otherwise specified (NOS). Based on H3K27me3 protein expression and chromosome 1q gain, all posterior fossa (PF) EPNs were classified as PF-A or PF-B. PD-L1 (SP263, Ventana) and CD8 immunohistochemistry was performed. The enrichment of RelA protein at the PDL1 promoter site was investigated using chromatin immunoprecipitation-qPCR (ChIP-qPCR) in three ST-RELA samples. A total of 83 intracranial EPNs were included in our preliminary data, and we discovered that PD-L1 expression correlates with increasing cytotoxic T-lymphocyte infiltrates in intracranial ependymomas using PD-L1 and CD8 IHC. The majority of PD-L1-positive intracranial EPNs are members of the ST-RELA subset, which also has the highest CTL density. Our findings suggest that ST-RELA EPNs could be used in immunotherapy to target immune checkpoint modulators. To further this research, we are looking into the presence of tumour infiltrating cells such as CD3, CD8, CD163, FOXP3, and others, which can aid in characterising the tumour microenvironment in cell types expressing PD-L1.This study will also be used to predict the prognostic significance of PD-L1/PD1 and other tumour cell infiltrating biomarkers in ependymomas and correlate it with survival outcomes.

RARE-30. Novel collision tumor of craniopharyngioma and ependymoma in a pediatric patient: a case study

Abstract Collision tumors are rare tumors comprised of two distinct histologies. In this case report, we discuss a suprasellar collision tumor consisting of adamantinomatous craniopharyngioma and supratentorial ependymoma in a pediatric patient. Case Presentation: Our patient was a two-year-old female with progressive craniopharyngioma status post cyst decompression with Ommaya reservoir placement, subcutaneous peginterferon, Ommaya taps, and subtotal resection. An MRI three months post-resection showed progression and treatment was started with subcutaneous interferon alfa. After eight weeks, she presented with new onset headaches and vomiting. MRI demonstrated tumor progression with associated obstructive hydrocephalus. She underwent a subtotal resection. Pathology revealed recurrent adamantinomatous craniopharyngioma and a 0.5cm ependymoma with classic histomorphology lacking anaplasia features. The ependymoma was positive for GFAP immunostain and EMA immunohistochemistry highlighted a ‘dot-like’ reaction. The Ki-67 proliferation index was very low (&amp;lt;1%). The limited diagnostic material precluded further genomic characterization of the ependymoma. The previous pathology was reviewed and no ependymoma was identified. Spine MRI was negative for metastatic disease. CSF cytology was negative for malignant cells. Following recovery from surgery, she received 54Gy (RBE) focal proton radiation. Eight months from completion of therapy, surveillance MRI shows stable residual tumor. Genetic work-up for cancer predisposition syndrome is in process despite no strong family history of cancer. Discussion: Due to the patient’s young age at diagnosis, our initial treatment strategy was to delay radiotherapy and utilize other treatment options. Following diagnosis with a collision tumor, the patient proceeded to radiotherapy to manage both tumor components. The role of interferon in the development of a collision tumor in this patient in unknown, but we suspect it to be unrelated. Conclusion: To our knowledge, this is the first documented case of a suprasellar collision tumor comprised of craniopharyngioma and ependymoma. Discovery of the collision tumor impacted the patient’s treatment plan.

ETMR-07. DNA methylation profiling of a series of rare CNS embryonal tumors in children: diagnostic and clinical impact

BACKGROUND: CNS embryonal tumors are a clinically and biologically heterogeneous group of tumors, more frequently arising in very young children. In the last few years, tumor classification through DNA methylation profiling has been demonstrated to be a powerful diagnostic tool which could be especially informative in this setting. METHODS: We reviewed original diagnosis and molecular profile of childhood CNS embryonal tumors other than medulloblastoma or AT/RT from a retrospective single-center cohort. Sixteen FFPE tissue samples from 14 unique patients (diagnosed from 1996 to 2017) were analyzed using DNA methylation arrays and matched with the Heidelberg classifier. Then, cohort characteristics and outcome were re-evaluated according to the results of the array. RESULTS: Median age at diagnosis was 2.7 years; there was no statistically significant difference between ETMRs and CNS embryonal tumors, NOS. Male to female ratio was 4:3. Median OS was 17,5 months (IQR 10.2-103.3 months) and ETMRs presented the worst outcome. Methylation profiling matched with an adequate score in 50% of samples (8/16). DNA methylation profile was consistent with ETMR in two samples but only one showed amplification of C19MC. Seven CNS embryonal tumors, NOS were properly reclassified as supratentorial ependymoma and diffuse pediatric-type HGG (4 and 1) or better defined as CNS neuroblastoma, FOXR2-altered (2). Methylation profiling added a unique diagnostic contribution in 64.3% of all cases (9/14). After the integration of methylation array results, survival markedly differed according to the novel integrated diagnoses; supratentorial ependymomas presented the longest median OS while no patients refined as CNS neuroblastoma or HGG survived. CONCLUSIONS: Our study confirmed that DNA methylation profiling provides relevant information for the classification of rare neoplasms like CNS embryonal tumors. Especially for selected cases with ambiguous histology, implementation of this tool should be considered to improve diagnostic precision and tailor patients’ management.

EPEN-10. Elucidating the biological difference between pediatric supratentorial ependymomas using genetically engineered brain tumor organoids.

Abstract Ependymomas (EPN) are neuroepithelial tumors of the central nervous system occurring both in adults and children. DNA methylation profiling has identified ten distinct molecular groups. In the supratentorial (ST) compartment, ZFTA-fusion positive and YAP1-fusion positive ST-EPN represent the two main groups in pediatric patients that are histologically very similar but have a completely different clinical behavior. While most ST-EPN-YAP1 patients survive, only 50% of ZFTA positive patients survive longer than 5 years. Improving survival of ZFTA positive patients requires better understanding of the tumor biology. Despite having different oncogenic fusions, it is unclear whether the cellular origin and tumor biology is related or not and why they respond differently to treatment. The development of human stem cell-derived brain organoids provides a novel opportunity to model these ST-EPN in vitro. These organoids mimic embryonal brain development, during which the cells-of-origin of ST-EPN as well as their micro-environment are present. To induce the development of ST-EPN in vitro, we genetically manipulated cerebral organoids to overexpress the group-specific oncogenic fusion proteins. Electroporating constructs expressing the different fusion proteins into organoids early in brain development (day 11), induced ectopic growth after approximately 30 days. Based on immunohistochemistry, we observed a different phenotype using either the ZFTA or YAP1 fusion constructs, as well as (over)expression of group-specific markers in the organoids. Preliminary transcriptomic analyses suggest that these genetically modified brain organoids represent models with features of human ST-EPN tumors. Ongoing single-cell RNA-sequencing and epigenomic analyses during normal organoid and tumor development will further reveal how well these models represent human primary tumors, how they develop in time, and possibly whether they originate from similar cells or different cells within these organoids. Hence, these models will uniquely contribute to the molecular and biological understanding of ependymomas and can be used to identify new targeted therapies.

EPEN-25. A novel spontaneous model of ZFTA-RELA fusion ependymoma

Ependymomas driven by the ZFTA-RELA fusion account for >70% of all supratentorial ependymomas. These tumours are now recognised in the WHO classification of CNS tumours and have been associated with a poor prognosis. Seven ZFTA-RELA fusion variants have been described: around two thirds of cases are fusion 1. No spontaneous genetically modified mouse models (GEMMS) have been described and current models require invasive intracranial injection (of transduced cells or RCAS-TVA system). Here we describe the first spontaneous GEMM of ZFTA-RELA fusion-driven ependymoma. Nestin-Flx-STOP-Flx-ZFTA-RELA (Fusion 1) or E1alpha-Flx-STOP-Flx-ZFTA-RELA open reading frames were targeted together with luciferase to the Rosa-26 locus. Breeding these mice with Nestin-CreERT2 or Blbp-Cre lines that drive recombination in neural progenitor cells resulted in forebrain tumours that could be tracked with bioluminescence imaging from P20. Tumours displayed NF-kB and L1CAM expression and ZFTA-RELA protein was detected using a novel in-house antibody. Tumours display expression of a known ZFTA-RELA fusion ependymoma transcriptomic signature. ZFTA-RELA tumours can be grown as neurospheres and passaged as allografts in nude mice. We provide the first spontaneous GEMM of this important group of ependymomas. We are now characterising these tumours histologically and transcriptomically relative to the human disease and using these to understand the lineage origins of ependymoma and plan use of conventional and novel treatments.

OTHR-46. Single institution experience using molecular analysis of pediatric CNS tumors

Molecular analysis of pediatric CNS tumors helps confirm the diagnosis, but can also guide treatment by identifying prognostic factors allowing for treatment stratification, and by unveiling active signaling pathways which can be targeted. This report is a retrospective review of the molecular analysis performed on all CNS tumors biopsied or resected at Children’s Minnesota over the last 3 years to evaluate our current practices. From 2019-2021, 118 patients with newly diagnosed CNS tumors underwent surgery followed by molecular assessment (14 IHC/FISH, 85 NGS, 7 methylation profiling) on 100% of medulloblastoma, other embryonal tumors, and schwannoma; 90% of ependymoma; 88% of HGG; 71% of LGG/glioneuronal/neuronal tumors; and 50% of meningioma and craniopharygioma. MAPK pathway alterations were seen in 84% of LGG/glioneuronal/neuronal tumors, with KIAA1540-BRAF fusion seen exclusively in pilocytic astrocytoma and BRAFV600E alterations seen in diffuse LGG (75%), PLNTY and PXA. Frequent alterations seen in HGG included H3F3A-K27M, H3F3A-G34, TP53, PDGFRA, ATRX, CDKN2A/B. Common gene alterations in medulloblastoma included monosomy 6 (100%) and alteration of CTNNB1 (50%) in WNT subgroup; PTCH1 (75%) in SHH subgroup; MYC/MYCN gain (60%) or amplification (60%) in Non-WNT/SHH subgroup. Alterations in FOXR2 in CNS neuroblastoma, SMARCB1 in ATRT and C19MC in ETMR confirmed these diagnoses. Supratentorial ependymoma showed ZFTQ-RELA fusion (100%) and infratentorial ependymoma showed chromosomal copy number changes including 1q gain (40%). Meningiomas showed deletion of NF2 and SMARCB1 and craniopharyngioma had alterations in CTNNB1. Molecular analysis confirmed the diagnosis in 23% of tumors, aided with targeted treatment in 20% of patients (82% of HGG, 9% of LGG, 13% of medulloblastomas) and allowed for risk-adapted treatment in 93% of medulloblastomas. These findings indicate that identification of pathogenic variants in CNS tumors aids in the diagnosis and treatment of pediatric CNS tumors and should be considered standard practice.

ZFTA (Zinc Finger Translocation Associated) Fusion in Supratentorial Ependymomas: Low Prevalence in South Asians and No Correlation with Survival

Supratentorial ependymomas (STEs) are an aggressive group of ependymomas, topographically distinct from their posterior fossa and spinal counterparts. Zinc finger translocation associated (ZFTA) fusion-positive cases have been reported to account for the majority of STEs, although data on its association with poorer outcomes are inconsistent. We assessed the prevalence of the ZFTA fusion by reverse-transcription polymerase chain reaction and fluorescence in situ hybridization in a cohort of 61 patients (68 samples) with STE. Our primary outcome was to determine the role of the ZFTA fusion on progression-free and overall survival of patients with STE. Our secondary objectives were to assess the impact of ZFTA fusion on nuclear factor (NF)-kB pathway signaling via surrogate markers of this pathway, namely COX-2, CCND1, and L1 cell adhesion molecule. ZFTA fusion was noted in 21.3% of STEs in our cohort. The presence of this rearrangement did not significantly impact the progression-free or overall survival of patients with STEs and was not associated with upregulation of markers of the NF-kB pathway. Only gross total resection was significantly associated with better progression-free survival. In contradiction to previous reports from across the world, the ZFTA fusion is far less prevalent among our population. It does not appear to drive NF-kB signaling or significantly affect outcomes. Gross total resection must be attempted in all cases of STE and adjuvant radiation and/or chemotherapy employed when gross total resection is not achieved.

Molecular subgrouping of ependymoma across three anatomic sites and their prognostic implications.

The 2021 WHO classification stratifies ependymoma (EPN) into nine molecular subgroups according to the anatomic locations which outperforms histological grading. We aimed at molecularly reclassifying 200 EPN using immunohistochemistry (IHC) and sequencing for ZFTA fusions in supratentorial (ST) EPN. Further, we assessed the utility of L1CAM, cyclinD1, and p65 markers in identifying ZFTA fusion. Demographic profiles, histologic features, molecular subgroups and clinical outcome were retrospectively analyzed. IHC for L1CAM, cyclinD1, p65, H3K27me3, and H3K27M and sequencing for ZFTA fusion were performed. ZFTA fusions were identified in 44.8% ST EPN. p65 displayed the highest specificity (93.8%), while L1CAM had the highest sensitivity (92.3%) in detecting ZFTA fusions. The negative predictive value approached 96.6% and sensitivity improved to 96.2% with combinatorial IHC (L1CAM, cyclinD1, p65). H3K27me3 loss (PF-A) was noted in 65% PF EPN. Our results provide evidence that a combination of two of three (L1CAM, p65, and cyclinD1) can be used as surrogate markers for predicting fusion. ZFTA fusion, and its surrogate markers in ST, and H3K27me3 and younger age (< 5years) in PF showed significant correlation with PFS and OS on univariate and Kaplan-Meier analysis. On multivariate analysis, H3K27me3 loss and younger age group are associated with poor clinical outcome.

Updates in the classification of ependymal neoplasms: The 2021 WHO Classification and beyond.

Ependymal neoplasms occur at all ages and encompass multiple tumor types and subtypes that develop in the supratentorial compartment, the posterior fossa, or the spinal cord. Clinically, ependymomas represent a very heterogeneous group of tumors from rather benign subependymomas to very aggressive and often deadly childhood ependymomas of the posterior fossa. Newly identified biological markers and classification schemes, e. g. based on global DNA methylation profiling, have led to the definition of 10 types of ependymal tumors and an improved prediction of patients’ outcome by applying the new classification system. While the exact genetic basis for several ependymoma types still remains unclear, the knowledge about ependymoma driving events has significantly increased within the last decade and contributed to a classification based on molecular characteristics and localization rather than histological features alone. Convincing evidence is now pointing towards gene fusions involving ZFTA or YAP1 causing the development of supratentorial ependymomas. Also, H3, EZHIP, or TERT mutations have been detected in a fraction of infratentorial ependymal tumors. Finally, MYCN amplifications have recently been identified in spinal ependymomas, in addition to the previously known mutations in NF2. This review summarizes how recent findings regarding biology, molecular tumor typing, and clinical outcome have impacted the classification of ependymomas as suggested by the updated 2021 WHO CNS tumor classification system. We focus on changes compared to the previous classification of 2016 and discuss how a formal grading could evolve in the future and guide clinicians to treat ependymoma patients.

Ependymoma associated protein Zfta is expressed in immature ependymal cells but is not essential for ependymal development in mice

The fusion protein of uncharacterised zinc finger translocation associated (ZFTA) and effector transcription factor of tumorigenic NF-κB signalling, RELA (ZFTA-RELA), is expressed in more than two-thirds of supratentorial ependymoma (ST-EPN-RELA), but ZFTA’s expression profile and functional analysis in multiciliated ependymal (E1) cells have not been examined. Here, we showed the mRNA expression of mouse Zfta peaks on embryonic day (E) 17.5 in the wholemount of the lateral walls of the lateral ventricle. Zfta was expressed in the nuclei of FoxJ1-positive immature E1 (pre-E1) cells in E18.5 mouse embryonic brain. Interestingly, the transcription factors promoting ciliogenesis (ciliary TFs) (e.g., multicilin) and ZFTA-RELA upregulated luciferase activity using a 5′ upstream sequence of ZFTA in cultured cells. Zftatm1/tm1 knock-in mice did not show developmental defects or abnormal fertility. In the Zftatm1/tm1 E1 cells, morphology, gene expression, ciliary beating frequency and ependymal flow were unaffected. These results suggest that Zfta is expressed in pre-E1 cells, possibly under the control of ciliary TFs, but is not essential for ependymal development or flow. This study sheds light on the mechanism of the ZFTA-RELA expression in the pathogenesis of ST-EPN-RELA: Ciliary TFs initiate ZFTA-RELA expression in pre-E1 cells, and ZFTA-RELA enhances its own expression using positive feedback.