Embryonal Tumor With Multilayered Rosettes Research Articles

A systematic review of the clinicopathological features and prognostic outcomes of DICER1-mutant malignant brain neoplasms.

DICER1-mutant malignant brain neoplasms are very rare tumors, and published data have relied on case reports or small case series. In this review, the authors aimed to systematically summarize the types and distribution patterns of DICER1 mutations, clinicopathological characteristics, and prognostic outcomes of these tumors. The authors searched PubMed and Web of Science for relevant studies. They included studies if they provided individual patient data of primary malignant brain tumors carrying DICER1 mutations. The authors found 16 studies consisting of 9 embryonal tumors with multilayered rosettes (ETMRs), 30 pineoblastomas, 52 primary intracranial sarcomas, and 27 pituitary blastomas. Pineoblastoma, ETMR, and pituitary blastoma were more likely to carry DICER1 germline mutations, while only a small subset of primary intracranial sarcomas harbored these mutations (p < 0.001). Nearly 80% of tumors with germline mutations also had another somatic mutation in DICER1. ETMR and primary intracranial sarcoma were associated with an increased risk for tumor progression and relapse compared with pituitary blastoma and pineoblastoma (p = 0.0025), but overall survival (OS) was not significantly different. Gross-total resection (GTR) and radiotherapy administration were associated with prolonged OS. ETMR, pineoblastoma, primary intracranial sarcoma, and pituitary blastoma should be considered rare phenotypes of the DICER1 syndrome, and families should be counseled and screened for associated tumors. ETMR and primary intracranial sarcoma had a higher risk of relapse. GTR and radiotherapy appeared to improve the OS of patients with DICER1-mutant malignant intracranial tumors.

Spectrum of paired-like homeobox 2b immunoexpression in pediatric brain tumors with embryonal morphology.

Paired‐like homeobox 2b (PHOX2B) is an established immunomarker for peripheral neuroblastoma and autonomic nervous system cells. We aimed to evaluate the utility of PHOX2B immunostaining in central nervous system (CNS) tumors with embryonal morphology. Fifty‐one tumors were stained with PHOX2B and submitted for whole slide image analysis: 35 CNS tumors with embryonal morphology (31 CNS embryonal tumors and four gliomas); and 16 peripheral neuroblastomas were included for comparison. Diffuse nuclear immunopositivity was observed in all (16/16) neuroblastomas (primary and metastatic). Among CNS embryonal tumors, focal immunoreactivity for PHOX2B was observed in most (5/7) embryonal tumors with multilayered rosettes (ETMR) and a single high‐grade neuroepithelial tumor (HGNET) with PLAGL2 amplification; the remaining 27 CNS tumors were essentially immunonegative (<0.05% positive). Among ETMR, PHOX2B expression was observed in a small overall proportion (0.04%–4.94%) of neoplastic cells but focally reached up to 39% in 1 mm ‘hot spot’ areas. In the PLAGL2‐amplified case, 0.09% of the total neoplastic population was immunoreactive, with 0.53% in the ‘hot spot’ area. Care should be taken in interpreting PHOX2B immunopositivity in a differential diagnosis that includes metastatic neuroblastoma and CNS tumors; focal or patchy expression should not be considered definitively diagnostic of metastatic peripheral neuroblastoma.

Abstract 1831: 9-ING-41, a glycogen synthase kinase 3B inhibitor targets TP53 in pediatric brain tumors ATRT and ETMR

Abstract Purpose: The study aims to determine the activity of the blood brain barrier permeable glycogen synthase kinase 3B (GSK3B) inhibitor, 9-ING-41, in pediatric brain tumors: embryonal tumor with multilayered rosettes (ETMR) and atypical rhabdoid tumor (ATRT). Methods: ETMR cell line, BT183, was grown in ultralow attachment flasks and ATRT cell lines: ATRT-7316-2187, ATRT-787197, and CHLA02 were grown in suspension flasks. Cells were treated with 9-ING-41 at dose range (0-20µM). After 24 and 48 hours of 9-ING-41 cells were analyzed by western blot. After 72 hours of 9-ING-41 cell viability was assessed with Cell Titer Glo 2.0 and IC50s were analyzed by GraphPad. RNA seq was performed on cells after 12, 24, and 48 hours of 9-ING-41 treatment. Total RNA was isolated with miRNeasy Mini Kit. RNA integrity was evaluated by an Agilent 2100 Bioanalyzer profile. rRNA was selectively depleted by employing RiboZero Globin. Next generation sequencing libraries were prepared using an Illumina TruSeq Stranded Total RNA kits. Read mapping was performed using STAR in BaseSpace. DESeq2 was used for normalization and differential expression analysis. Ingenuity Pathway Analysis was performed. Results: The IC50s of 9-ING-41 in the cell lines BT183, CHLA02-ATRT, ATRT-787197, and ATRT-2187 are 145.5nM, 481.2nM, 503.1nM, and 528.3nM, respectively. In BT183 cells treated with 150nM 9-ING-41 for 48 hours, XIAP decreased significantly by 55% and cleaved caspase 3 increased significantly by 29-fold, quantified by densitometry. In addition, western blot showed a decrease in LIN28A and an increase in TP53 protein levels. RNA-seq data show an increase in the p53 signaling pathway in the ETMR BT183 cell line with z-scores of 2.53 and 3 (p=3.73x10-13 and 2.35x10-14) after 12 and 24 hours of 150nM 9-ING-41, respectively. In ATRT-787197 cells p53 signaling increased with z-scores of 1.0 and 2.236 (p=2.62x10-8 and 2.89x10-5) after 24 and 48 hours of 400nM 9-ING-41, respectively. RNA seq data also show the ability of 9-ING-41 to significantly increase the expression of CDK inhibitor, CDKN1A, and pro-apoptotic genes BBC3, DRAM1, and BAX in ETMR and ATRT while significantly decreasing stemness associated genes: SMO and GLI1 in ETMR. Conclusions: 9-ING-41 decreases ETMR and ATRT cell viability at clinically relevant concentrations and these doses increase the p53 pathway and induce apoptosis while reducing the expression of stemness associated genes. Citation Format: Jason L. Haw, Elizabeth M. Moore, Kimberly McKinney, Karl Dykema, Abhinav Nagulapally, Nury Steuerwald, Giselle S. Sholler. 9-ING-41, a glycogen synthase kinase 3B inhibitor targets TP53 in pediatric brain tumors ATRT and ETMR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1831.

Germ cell tumors with neuroglial differentiation do not show molecular features akin to their central nervous system counterpart: experience from extra-gynecological sites.

Teratomas with secondary somatic malignancy showing neuroglial differentiation (central nervous system (CNS)-type tumors) arising from a glial or neuroepithelial component is a very uncommon event and primarily described in the ovary. We aimed to describe the morphological spectrum and molecular features of CNS type of neuroepithelial tumors arising from the germ cell tumors (GCT) in the extra-gynecological sites. All cases of teratoma and mixed GCT arising from the non-gynecological sites over 7years were screened for CNS type of neuroepithelial tumors. Detailed histological and immunohistochemical analysis was performed. IDH1 and 2 sequencings were performed in the glial tumors. Fluorescent in situ hybridization (FISH) was performed for EWSR1 rearrangement, 19/19q co-deletion, CDKN2A homozygous deletion, EGFR amplification, and C19MC amplification, wherever required. Out of 302 GCTs examined, the neuroglial tumor was detected in 15 cases. It included nine cases of glial tumors (including one pilocytic astrocytoma (grade I), two diffuse astrocytomas (grade II), one oligodendroglioma (grade II), one gemistocytic astrocytoma (grade II), three anaplastic astrocytomas (grade III), and one case of glioblastoma (grade IV)) and six cases of the embryonal tumor with multilayered rosettes (ETMR). None of the gliomas showed IDH mutation by immunohistochemistry or sequencing. The ETMR cases did not show Lin28 expression or C19MC amplification. To conclude, the spectrum of neuroglial tumors arising from teratoma in the extragonadal sites is vast and most commonly includes glial neoplasms and embryonal tumors. Our findings indicate that the genotype and pathogenesis of tumors with neuroglial differentiation in teratoma are distinct from their CNS counterpart.

ETMR-05. Single-cell transcriptomics of ETMR reveals developmental cellular programs and tumor-pericyte communications in the microenvironment

BACKGROUND: Embryonal tumors with multilayered rosettes (ETMR) are pediatric brain tumors bearing a grim prognosis, despite intensive multimodal therapeutic approaches. Insights into cellular heterogeneity and cellular communication of tumor cells with cells of the tumor microenvironment (TME), by applying single-cell (sc) techniques, potentially identify mechanisms of therapy resistance and target-directed treatment approaches. MATERIAL AND METHODS: To explore ETMR cell diversity, we used single-cell RNA sequencing (scRNA-seq) in human (n=2) and murine ETMR (transgenic mode; n=4) samples, spatial transcriptomics, 2D and 3D cultures (including co-cultures with TME cells), multiplex immunohistochemistry and drug screens. RESULTS: ETMR microenvironment is composed of tumor and non-tumor cell types. The ETMR malignant compartment harbour cells representing distinct transcriptional metaprograms, (NSC-like, NProg-like and Neuroblast-like), mirroring embryonic neurogenic cell states and fuelled by neurogenic pathways (WNT, SHH, Hippo). The ETMR TME is composed of oligodendrocyte and neuronal progenitor cells, neuroblasts, microglia, and pericytes. Tumor-specific ligand-receptor interaction analysis showed enrichment of intercellular communication between NProg-like ETMR cells and pericytes (PC). Functional network analyses reveal ETMR-PC interactions related to stem-cell signalling and extracellular matrix (ECM) organization, involving factors of the WNT, BMP, and CxCl12 networks. Results from ETMR-PC co-culture and spatial transcriptomics pointed to a pivotal role of pericytes in keeping ETMR in a germinal neurogenic state, enriched in stem-cell signalling. Drug screening considering cellular heterogeneity and cellular communication suggested novel therapeutic approaches. CONCLUSION: ETMR demonstrated diversity in the microenvironment, with enrichment of cell-cell communications with pericytes, supporting stem-cell signalling and interfering in the organization of the tumor extracellular matrix. Targeting ETMR-PC interactions might bring new opportunities for target-directed therapy.

TBIO-05. R-loops in pediatric brain tumors

Abstract R-loops are structures containing DNA-RNA hybrids and displaced single-stranded DNA, which are widely distributed across genomic regions. The generation and removal of R-loops is dynamically regulated by several factors including helicases and topoisomerases. Previously, we have identified high levels of R-loops associated with genomic instability in Embryonal Tumors with Multilayered Rosettes (ETMR) (Lambo et al. 2019), which is associated with a sensitivity to drugs targeting topoisomerases and DNA repair. However, it is unknown whether there are other pediatric cancers with high levels of R-loops, and whether R-loops in these tumors are also associated with genomic instability and sensitivity to specific treatments. Here, we have used real R-loops sequencing data sets, including ETMR DRIP-seq data, and the bioinformatic tool QmRLFS-finder (Wongsurawat et al. 2012), which can predict R-loops forming sequences (RLFSs) based on DNA sequence composition. By performing bioinformatic analyses of DRIP-seq data sets and integrating these with RLFSs from human reference genome simulations, we aim to analyze how reliable the different methods are to measure and predict R-loops and what role they have in cancer genomes. In ETMR, R-loops signals from DRIP-seq data were enriched around transcriptional start sites (TSS), comparable to R-loops distribution and enrichment in other published cell models. However, the overlap with RLFSs was limited, indicating that more validations with real DRIP-seq data from more ETMRs and other pediatric cancers are needed to validate the different methods. Taken together, until now, we developed methods to (semi-)quantify R-loops by combining real sequencing data and computational simulation approaches to investigate R-loops distribution in cancer genomes and to investigate whether R-loops are associated with breakpoints or other genetic aberrations in pediatric cancer entities.

RONC-16. Proton therapy for patients with embryonal tumor with multilayered rosettes in early childhood – results of the prospective KiProReg Study

Abstract OBJECTIVES: Embryonal tumors with multilayered rosettes (ETMR) are rare malignancies of the central nervous system occurring predominantly in early childhood. Little is known about optimal time point and target volume of radiotherapy for the respective patients. The aim of this analysis was to evaluate treatment outcome in pediatric patients with ETMR treated with Proton Therapy (PT). METHODS: Between May 2016 and August 2021, 15 patients (9 male /6 female) with ETMR received PT in our institution and were enrolled in the prospective registry study KiProReg. Patient characteristics, treatment and outcome according to standardized follow-up data were descriptively analyzed by summarizing frequencies due to small sample size. RESULTS: Median age at PT was 3.0 years (range, 1.6-4.0 years). Three patients presented with metastatic disease (M1 n=1; M2 n=1; M2/3 n=1). Eight patients were treated with salvage PT at tumor progression or recurrence. Residual disease was present in three patients at start of PT. Tumor site was infratentorial (n=3) or supratentorial (n=12). All patients received Chemotherapy (CTX) prior to PT including high dose CTX (n=9) and intrathecal CTX (n=3). Concomitant CTX with temozolomide was administered in one patient. Patients received local PT (n=8) or craniospinal irradiation (CSI) followed by a local boost (n=7). Median dose was 54.0 Gy(RBE) (range, 3.6 -59.4 Gy(RBE)). PT was terminated prematurely in one patient due to cerebral edema and disease progression. Median follow-up after diagnosis was 16.3 months (range, 6.6-65.7 months) and 5.1 months (range, 0-62.4 months) after PT. Four patients treated for salvage experienced disease progression within three months after PT, three of them deceased. CONCLUSION: Preliminary results suggest promising outcomes for childhood ETMR after PT, especially in patients treated at initial diagnosis. Longer follow-up and larger cohorts are desirable to assess long-term survival and necessity of CSI.

ETMR-10. Retrospective molecular re-evaluation of CNS PNETs; a population-based study

Abstract BACKGROUND: The heterogeneous group of tumors, primitive neuro-ectodermal tumors of the central nervous system (CNS-PNETs), is a group of rare childhood embryonal tumors associated with a poor prognosis. In recent years, molecular analyzes have shown that CNS-PNETs consist of high-grade gliomas (HGG), ependymomas, different embryonal entities like atypical teratoid /rhabdoid tumors (AT/RT), CNS neuroblastoma FOXR2 and embryonal tumor with multi-layered rosettes (ETMR). Each of these tumor types is unusual and long-term clinical follow-up data are sparse. METHODS: We retrospectively re-evaluated all children (0-18 years old) diagnosed with a CNS-PNET in Sweden during 1984-2015. In total, 88 supratentorial CNS-PNETs were identified in the Swedish Childhood Cancer Registry and from these formalin-fixed paraffin-embedded tumor material was available for 69 patients. All tumors were reviewed histopathologically by an experienced neuropathologist and were analyzed using genome-wide DNA methylation profiling and classified by the MNP brain tumor classifier. RESULTS: The largest entities, after re-evaluation, were HGG (30%), CNS NB-FOXR2 (12%), AT/RT (10%) and ETMR (8%). Some tumors were difficult to classify and will be further evaluated molecularly. Some examples: Best treatment results were seen for patients with CNS-NB FOXR2 (5-year PFS: 100%) where all patients had received craniospinal radiotherapy (CSI). Patients with ETMR were all very young and survival data show early progression and poor survival (5-year OS 34%). CONCLUSIONS: Although the patient material is relatively small, it is population-based with long follow-up times. Our findings are in line with other studies and shows that CSI is important for cure for CNS-NB FOXR2 and that intensive multi-modal therapies needs to be evaluated in up-front studies for these rare embryonal tumors.

ETMR-04. Embryonal tumor with multi-layered rosettes (ETMR) located in the brainstem: a case report on clinical decision-making and a multimodal, interdisciplinary treatment approach including interstitial brachytherapy

OBJECTIVE: Embryonal tumors with multi-layered rosettes (ETMR) (CNS WHO grade 4) comprise a rare and malignant tumor type affecting predominantly infants below 3 years of age. The treatment consists of maximal safe surgical resection, irradiation, and intensive medulloblastoma type chemotherapy. Despite aggressive treatment, the prognosis of these patients remains poor, especially for brainstem tumors. We present the case of a male infant diagnosed with a brainstem ETMR, successfully treated with an interdisciplinary multimodal approach, including stereotactic interstitial brachytherapy. RESULTS: A 19 month old boy first presented with hemiparesis, intermittent bradycardia and reduced consciousness. Initial imaging showed a brainstem lesion with characteristic features of a diffuse intrinsic pontine glioma (DIPG). We performed stereotactic biopsy to confirm the diagnosis and initiated temozolomide treatment. While the pathology result was still pending, the boy’s clinical condition deteriorated to a soporic state with stretch synergisms. By emergency open surgery, partial resection was achieved. Eventually, the patient recovered rapidly. After the diagnosis of ETMR was established, medulloblastoma type chemotherapy (systemic carboplatin/etoposide; intrathecal methotrexate) was administered. After two cycles, the patient showed only residual right-sided hemiparesis. However, imaging demonstrated only a minimal reduction of the tumor size. Therefore, stereotactic interstitial brachytherapy using 125iodine seeds and subsequent high-dose chemotherapy (HDCT) with autologous stem cell transplantation (ASCT) was performed and overall tolerated well. After two months of brachytherapy and two additional cycles of chemotherapy, the MRI showed ≥ 50% reduction in tumor volume and no neurological deficit can be clinically detected. CONCLUSION: This case indicates that stereotactic interstitial brachytherapy during intensive systemic chemotherapy is feasible. It may provide a suitable treatment for malignant infant brain tumors. Furthermore, it shows that paediatric patients are capable of recovery even after devastating neurological symptoms. Lastly, it emphasizes the importance of multidisciplinary and multimodal treatment for rare diseases.

ETMR-09.In vitro modelling of embryonal tumors with multilayered rosettes (ETMR) and other novel brain tumor types.

Over the last decade, molecular characterization has resulted in many tumors previously classified as central nervous system primitive neuroectodermal tumors (CNS-PNETs) now being classified into their own distinct tumor types. These novel types are often characterized by very specific genomic aberrations. For instance, embryonal tumors with multilayered rosettes (ETMR) harbor amplifications of miRNA cluster C19MC or complex DICER1 mutations, while in CNS neuroblastoma with FOXR2 activation structural aberrations result in aberrant FOXR2 expression. Despite the presence of distinct oncodrivers, our understanding of these tumors is still limited. To elucidate tumor biology and to discover tumor specific treatments, we need to uncover how these oncodrivers contribute to tumorigenesis. However, a bottleneck in basic and translational research of these novel tumor types, is the lack of representative preclinical models, especially in vitro. To overcome this hurdle, we aim to mimic tumor development in genetically modified brain organoids. Human brain organoids derived from pluripotent stem cells are generated to represent either the developing forebrain or cerebellum. To mimic oncodriving events, DNA plasmids are introduced via electroporation into the proposed cell-of-origin populations to knockout tumor suppressor genes or overexpress oncogenes. By detecting fluorescent proteins encoded by the plasmids, electroporated cells are followed over time. Based on our preliminary data, for instance, overexpression of C19MC results in ectopic expansion of the electroporated cells. Ongoing histological and molecular characterizations, including (single cell) transcriptomic and epigenomic analyses, will reveal to which extend these organoid models resemble the specific human tumor types. Although further validation is required, these organoid models provide a novel avenue to study especially brain tumor types with distinct oncodriving events for which patient-derived models have not yet been established. They also allow for in-depth analyses of the potential cells of origin and the contribution of different mutations to tumor biology.

ETMR-16. Embryonal tumors with multi-layered rosettes: A case series in treatment for newly diagnosed children

Abstract BACKGROUND: Embryonal tumors with multi-layered rosettes (ETMRs) are rare pediatric brain tumors with poorly defined prognostic features, standard of care treatments or outcome data. Recent data suggest that high-dose chemotherapy and radiotherapy is correlated with improved survival compared to chemotherapy alone. CASE DESCRIPTIONS: Four patients with newly diagnosed ETMRs were treated with 2 cycles of induction chemotherapy per PBTC-026 using isotretinoin, vorinostat, vincristine, cisplatin, etoposide, cyclophosphamide with added intrathecal topotecan. Second look surgery was performed if not in complete remission (CR). Consolidation was with three cycles of marrow-ablative chemotherapy (carboplatin and thiotepa) with autologous hematopoietic cell rescue followed by focal irradiation and 12 cycles of maintenance chemotherapy with intrathecal topotecan, vorinostat and isotretinoin. Patient 1 was a 3-year-old female with right parietal tumor, localized, and achieved gross total resection (GTR). Patient 2 was an 11-month-old male with posterior fossa tumor, localized, and achieved subtotal resection. Patient 3 was a 9-month-old female with posterior fossa tumor with near GTR, and metastasis to T12/L1 and L3/L4. [DTB1] [HNT2] Patient 4 was a 34-month-old male with a right frontal lobe tumor, localized and achieved GTR. Patient 1 is now 18 months from diagnosis and in CR. Patient 2 had second look surgery both after induction and consolidation but suffered neurologic injury to the brainstem which led family to decline further therapy. He is currently 14 months from diagnosis with stable residual disease. Patient 3 had local disease recurrence following radiation therapy at 10 months post diagnosis[DTB3] [HNT4] , prior to maintenance. Patient 4 remains in CR at 13 months from diagnosis, currently in maintenance with vorinostat and isotretinoin but declined intrathecal topotecan. CONCLUSION: This case series adds to current knowledge of intensive multi-modal therapy for newly diagnosed ETMR. Further study to define standard optimal treatments in this high-risk group of patients is warranted.

MODL-10. Tumor-brain-organoids as a model for pediatric brain tumors research

Abstract BACKGROUND: Embryonal brain neoplasms like atypical teratoid rhabdoid tumor (ATRT) or embryonal tumor with multilayered rosettes (ETMR) still have a very poor outcome despite intensive treatment including chemotherapy, irradiation and surgery. To date, precision oncology has identified clinically relevant innovative therapeutic targets only for a minor subpopulation of pediatric brain tumor patients, which may be due to current in vitro screens not recapitulating the cellular heterogeneity and cellular interactions in vivo. As cellular heterogeneity and cellular interactions majorly influence the response of tumor cells to treatment, we established an innovative 3D screening platform that combines human neural tissue surrounding primary tumor tissue. METHODS: We established a model of tumor-brain-organoids (TBO) by incorporating embryonal tumor cells (ATRT and ETMR tumor cells) into hiPSC-derived forebrain organoids. Using whole mount immunostaining (WMI), we evaluated cancer-phenotype, the neuronal and progenitor cell distribution in brain organoids, and we performed drug screening analysis. Furthermore, we used single-cell RNA-sequencing to characterize the cellular heterogeneity and the effect of tumor-organoid cell-cell communication on transcriptional programs. RESULTS: ATRT as well as ETMR tumor cells incorporated extensively into the organoid tissue. We observed remarkable differences in the invasiveness of ATRT-MYC cells into TBO in comparison with ATRT-SHH and ETMR cells via high content imaging. Moreover, tumor cells affected the gene expression of different cell types of the organoid by upregulating genes of important signaling/growth related pathways (e. g. MAP2K2, IGFBP2) and epigenetic regulators (like BRD7). Screening through a 300 compound FDA-approved drug library in these TBO, we identified potential innovative therapeutic approaches against these embryonal tumors. CONCLUSION: Tumor-brain-organoids can be used as a platform to study tumor biology, tumor interactions with its neural tissue microenvironment, as well as for high-throughput drug and toxicity screening in pediatric brain tumor precision oncology.

MODL-01. Targeting replication stress in pediatric brain tumors

Previously, we have found that Embryonal Tumors with Multilayered Rosettes (ETMR) tumor cells harboring high levels of R-loops, a potential marker for replication stress and genomic instability, are vulnerable to a combination of topoisomerase and PARP inhibitors. To follow up on this, we investigated whether other pediatric brain tumor types with high levels of R-loops, such as MYC-amplified Group 3 medulloblastoma (MB) and ZFTA-fusion positive ependymoma, are also sensitive to these inhibitors. First, we performed in vitro drug screens using HD-MB03, a Group 3 MB cell line, and the ETMR cell line BT183, and in both screens PARP inhibitors were identified as the most synergistic combination partners for the topoisomerase inhibitor Irinotecan, respectively the active metabolite SN-38. Normal Astrocytes were not sensitive to these combinations. Secondly, we performed in vivo studies using patient-derived xenograft (PDX) models injected subcutaneously or intracranially into NSG mice, and treated with the PARP inhibitor Pamiparib, Irinotecan or a combination of both. For a MYC-amplified Group 3 MB and a ZFTA-fusion positive Ependymoma model, both injected intracranially, treatment with Irinotecan or the combination led to a significant survival benefit and inhibition of tumor growth including transient tumor shrinkage, but addition of Pamiparib did not add any further benefit in vivo, even though intratumoral PARP was inhibited by at least 80%. In contrast, in the subcutaneously injected ETMR model, the combination treatment with Irinotecan and Pamiparib led to a synergistic effect and complete regression of the tumors. Further refinements of the treatment strategy as dose adaptations and the use of a pegylated version of SN-38 (PLX038A) did also not induce a synergistic effect of the drugs for the intracranial tumors. Additional in vivo studies to evaluate the differences in efficacy and whether these are tumor specific or due to incomplete brain penetrance of the drugs are ongoing.

ETMR-02. Overexpression of Lin28A in neural progenitor cells in vivo does not lead to brain tumor formation but results in reduced spine density

The RNA binding protein LIN28A is a stem- and progenitor marker and one of the factors necessary to induce pluripotent stem cells. An overexpression of LIN28A has been identified in malignant brain tumors called embryonal tumors with multilayered rosettes (ETMR) but its specific role during brain development remains largely unknown. Radial glia cells of the ventricular zone (VZ) are proposed as a cell of origin for ETMR. We asked whether an overexpression of LIN28A in such cells might affect brain development or result in the formation of brain tumors. Constitutive overexpression of LIN28A in hGFAP-cre::lsl-Lin28A (GL) mice led to a transient increase of proliferation in the cortical VZ at embryonic stages but no postnatal brain tumor formation. Postnatally, GL mice displayed a pyramidal cell layer dispersion of the hippocampus and altered spine and dendrite morphology, including reduced dendritic spine densities in the hippocampus and cortex. GL mice displayed hyperkinetic activity and differential quantitative MS-based proteomics revealed altered time dependent molecular functions regarding mRNA processing and spine morphogenesis. Phosphoproteomic analyses indicated a downregulation of mTOR pathway modulated proteins such as Map1b being involved in microtubule dynamics within a crosstalk of Gsk3b/Rho-Rac/Map1b signaling. In conclusion, we show that Lin28A overexpression transiently increases proliferation of neural precursor cells but it is not sufficient to drive brain tumors in vivo. In contrast, Lin28A impacts on protein abundancy patterns related to spine morphogenesis and phosphorylation levels of proteins involved in microtubule dynamics, resulting in decreased spine densities of neurons in the hippocampus and cortex as well as in altered behavior. Our work provides new insights into the role of LIN28A for neuronal morphogenesis and development and may reveal future targets for treatment of ETMR patients.

Embryonal tumors in the WHO CNS5 classification: A Review.

Embryonal tumors are a heterogenous group of neoplasms mostly defined by recurrent genetic driver events. They have been, previously, broadly classified as either medulloblastoma or supratentorial primitive neuroectodermal tumors (PNETs). However, the application of DNA methylation/gene expression profiling in large series of neoplasms histologically defined as PNET, revealed tumors, which showed genetic events associated with glial tumors. These findings led to the definitive removal of the term "PNET" in the 2016 World Health Organization (WHO) classification of CNS tumors. Moreover, further studies on a large scale of methylation profiling have allowed the identification of new molecular-defined entities and have largely influenced the 5th edition of the WHO classification of CNS tumors (WHO CNS5) for both medulloblastomas and other CNS embryonal tumors. The importance of molecular characteristics in CNS embryonal tumors is well represented by the identification of different molecular groups and subgroups in medulloblastoma. So, in the CNS5, the emerged group 3 and group 4 belong to the classification, and the four molecular and morphologic types are now combined into a unique section. Among other embryonal tumors, two new recognized entities are introduced in CNS5: CNS neuroblastoma, FOXR2-activated, and CNS tumor with BCOR internal tandem duplication (ITD). Embryonal tumor with multilayered rosettes (ETMR), already present in the previous classification now has a revised nomenclature as a result of the new DICER1 alteration, additional to the formerly known C19MC. Regarding atypical teratoid/rhabdoid tumor (AT/RT), three molecular subgroups are recognized in CNS5. The combination of histopathological and molecular features reflects the complexity of all these tumors and gives critical information in terms of prognosis and therapy. This encourages the use of a layered diagnostic report with the integrated diagnosis at the top, succeeded by layers including the histological, molecular, and other essential details.

Two cases of Embryonal tumor with Multilayered Rosettes (ETMRs) treated with high-dose chemotherapy and bevacizumab combined with dose escalation radiotherapy

Two cases of Embryonal tumor with Multilayered Rosettes (ETMRs) treated with high-dose chemotherapy and bevacizumab combined with dose escalation radiotherapy

Embryonal tumor with multilayered rosettes; rare pediatric CNS tumor. A case report and review of literature

Embryonal tumor with multilayered rosettes (ETMR), C19MC-altered is a newly designated entity of the embryonal tumors of the central nervous system (CNS) according to the 2016 WHO classification system of CNS. Characteristically, these tumors are newly defined based on their specific molecular genetic amplification in chromosome 19q13.42 found at locus C19MC. To the best of our knowledge, we present the first reported case of ETMR in Saudi Arabian pediatric population. A 2-year-old boy presented to the hospital with generalized tonic-colonic seizure, vomiting, irritability, and inability to walk. Computed tomography (CT) scan showed a large left thalamic supratentorial brain tumor. The tumor measured 6.1 × 5.6 × 5.6 cm and was characterized by cystic changes, prominent vasculature, and calcifications. Histopathology, immunohistochemistry examination, and fluorescence in situ hybridization (FISH) analysis confirmed the diagnosis of ETMR. In addition to reporting this rare case, we provide a brief literature review, treatment options, patient outcome, and disease prognosis.

Co-activation of Sonic hedgehog and Wnt signaling in murine retinal precursor cells drives ocular lesions with features of intraocular medulloepithelioma

Intraocular medulloepithelioma (IO-MEPL) is a rare embryonal ocular neoplasm, prevalently occurring in children. IO-MEPLs share histomorphological features with CNS embryonal tumors with multilayered rosettes (ETMRs), referred to as intracranial medulloepitheliomas. While Sonic hedgehog (SHH) and WNT signaling pathways are crucial for ETMR pathogenesis, the impact of these pathways on human IO-MEPL development is unclear. Gene expression analyses of human embryonal tumor samples revealed similar gene expression patterns and significant overrepresentation of SHH and WNT target genes in both IO-MEPL and ETMR. In order to unravel the function of Shh and Wnt signaling for IO-MEPL pathogenesis in vivo, both pathways were activated in retinal precursor cells in a time point specific manner. Shh and Wnt co-activation in early Sox2- or Rax-expressing precursor cells resulted in infiltrative ocular lesions that displayed extraretinal expansion. Histomorphological, immunohistochemical, and molecular features showed a strong concordance with human IO-MEPL. We demonstrate a relevant role of WNT and SHH signaling in IO-MEPL and report the first mouse model to generate tumor-like lesions with features of IO-MEPL. The presented data may be fundamental for comprehending IO-MEPL initiation and developing targeted therapeutic approaches.

Therapeutic implications of improved molecular diagnostics for rare CNS embryonal tumor entities: results of an international, retrospective study.

Only few data are available on treatment-associated behavior of distinct rare CNS embryonal tumor entities previously treated as "CNS-primitive neuroectodermal tumors" (CNS-PNET). Respective data on specific entities, including CNS neuroblastoma, FOXR2 activated (CNS NB-FOXR2), and embryonal tumors with multilayered rosettes (ETMR) are needed for development of differentiated treatment strategies. Within this retrospective, international study, tumor samples of clinically well-annotated patients with the original diagnosis of CNS-PNET were analyzed using DNA methylation arrays (n = 307). Additional cases (n = 66) with DNA methylation pattern of CNS NB-FOXR2 were included irrespective of initial histological diagnosis. Pooled clinical data (n = 292) were descriptively analyzed. DNA methylation profiling of "CNS-PNET" classified 58 (19%) cases as ETMR, 57 (19%) as high-grade glioma (HGG), 36 (12%) as CNS NB-FOXR2, and 89(29%) cases were classified into 18 other entities. Sixty-seven (22%) cases did not show DNA methylation patterns similar to established CNS tumor reference classes. Best treatment results were achieved for CNS NB-FOXR2 patients (5-year PFS: 63% ± 7%, OS: 85% ± 5%, n = 63), with 35/42 progression-free survivors after upfront craniospinal irradiation (CSI) and chemotherapy. The worst outcome was seen for ETMR and HGG patients with 5-year PFS of 18% ± 6% and 22% ± 7%, and 5-year OS of 24% ± 6% and 25% ± 7%, respectively. The historically reported poor outcome of CNS-PNET patients becomes highly variable when tumors are molecularly classified based on DNA methylation profiling. Patients with CNS NB-FOXR2 responded well to current treatments and a standard-risk CSI-based regimen may be prospectively evaluated. The poor outcome of ETMR across applied treatment strategies substantiates the necessity for evaluation of novel treatments.

IMMU-14. COMPUTATIONAL DECONVOLUTION OF TUMOR-INFILTRATING IMMUNE COMPONENTS IN PEDIATRIC NERVOUS SYSTEM TUMORS

IntroductionIn the last decade, checkpoint inhibitor-based immunotherapy has been a groundbreaking development in the treatment of cancer. However, only a subset of patients treated with immune checkpoint inhibitors show long-lasting clinical benefit. Studies showed the tumor immune microenvironment (TME) as a particularly important factor influencing treatment response, critical for the design of other or combinatorial immunotherapy treatment strategies. Extensive research has been performed in the adult cancer field to unravel its immunogenomic aspects. However, in pediatric cancer this insight into tumor-infiltrating immune components is still lacking. This study aims to provide insight into the landscape of the immune microenvironment in pediatric primary nervous system tumors. MethodsBulk RNA-seq data of 936 pediatric primary solid tumors acquired from multiple international initiatives including Therapeutically Applicable Research To Generate Effective Treatments (TARGET), the International Cancer Genome Consortium (ICGC) and the Children’s Brain Tumor Tissue Consortium (CBTTC) were included in this study. We applied computational tumor immune microenvironment deconvolution, repurposed RNA-seq data to recover infiltrating T- and B-cell clonotypes and studied checkpoint gene expression across pediatric neural tumors. ResultsAmong pediatric neural tumors, embryonal tumors with multilayered rosettes (ETMR) and medulloblastomas (MB) were least immune infiltrated. Neuroblastomas (NBL) had the highest T-cell infiltration among pediatric cancers, while atypical teratoid/rhabdoid tumors (ATRT) had the highest levels of CD8 T cell infiltration among pediatric CNS tumors. While tumor mutational burden (TMB) was associated with immune cell infiltration in adult lung cancers and melanomas, we found no significant associations in pediatric cancers. The majority of NBL samples expressed LAG3, but ~10% of samples had elevated levels of TIM3 gene expression, suggesting a distinct mode of immunosuppression in this subset.