BCOR Internal Tandem Duplication Research Articles

Recurrent and novel fusions detected by targeted RNA sequencing as part of the diagnostic workflow of soft tissue and bone tumours.

The identification of gene fusions has become an integral part of soft tissue and bone tumour diagnosis. We investigated the added value of targeted RNA-based sequencing (targeted RNA-seq, Archer FusionPlex) to our current molecular diagnostic workflow of these tumours, which is based on fluorescence in situ hybridisation (FISH) for the detection of gene fusions using 25 probes. In a series of 131 diagnostic samples targeted RNA-seq identified a gene fusion, BCOR internal tandem duplication or ALK deletion in 47 cases (35.9%). For 74 cases, encompassing 137 FISH analyses, concordance between FISH and targeted RNA-seq was evaluated. A positive or negative FISH result was confirmed by targeted RNA-seq in 27 out of 49 (55.1%) and 81 out of 88 (92.0%) analyses, respectively. While negative concordance was high, targeted RNA-seq identified a canonical gene fusion in seven cases despite a negative FISH result. The 22 discordant FISH-positive analyses showed a lower percentage of rearrangement-positive nuclei (range 15-41%) compared to the concordant FISH-positive analyses (>41% of nuclei in 88.9% of cases). Six FISH analyses (in four cases) were finally considered false positive based on histological and targeted RNA-seq findings. For the EWSR1 FISH probe, we observed a gene-dependent disparity (p = 0.0020), with 8 out of 35 cases showing a discordance between FISH and targeted RNA-seq (22.9%). This study demonstrates an added value of targeted RNA-seq to our current diagnostic workflow of soft tissue and bone tumours in 19 out of 131 cases (14.5%), which we categorised as altered diagnosis (3 cases), added precision (6 cases), or augmented spectrum (10 cases). In the latter subgroup, four novel fusion transcripts were found for which the clinical relevance remains unclear: NAB2::NCOA2, YAP1::NUTM2B, HSPA8::BRAF, and PDE2A::PLAG1. Overall, targeted RNA-seq has proven extremely valuable in the diagnostic workflow of soft tissue and bone tumours.

Primitive myxoid mesenchymal tumor of infancy, an enigmatic entity: A case report.

Primitive myxoid mesenchymal tumor of infancy (PMMTI) is a rare soft tissue neoplasm of intermediate malignant potential occurring predominantly in infants and younger children. It can show aggressive local infiltration of surrounding structures and frequent relapses but rare metastasis. Morphologically, it is characterized by a proliferation of immature mesenchymal cells in a myxoid background with mild cytologic atypia and branching vessels. The tumor cells demonstrate immunoreactivity with vimentin and BCOR, however, are negative for more specific lineage markers. Its molecular hallmark is BCOR-internal tandem duplication. Previously classified as infantile fibrosarcoma/undifferentiated sarcoma, the association with BCOR-ITD has led to its reclassification as part of a distinct entity-"Sarcoma with BCOR Genetic Alterations"-in the 2020 WHO Classification of Soft Tissue Tumours. Here is a report of a case of a 2-year-old boy with a recurrent scalp mass identified as a primitive myxoid mesenchymal tumor of infancy.

CNS tumor with CREBBP::BCORL1 Fusion and pathogenic mutations in BCOR and CREBBP: expanding the spectrum of BCOR-altered tumors

The fifth edition of the World Health Organization (WHO) classification of central nervous system (CNS) tumors introduced the new tumor type CNS tumor with BCOR internal tandem duplication (ITD), characterized by a distinct DNA methylation profile and peculiar histopathological features, including a circumscribed growth pattern, ependymoma-like perivascular pseudorosettes, microcystic pattern, absent or focal GFAP immunostaining, OLIG2 positivity, and BCOR immunoreactivity. We describe a rare case of a CNS tumor in a 45-year-old man with histopathological and immunohistochemical features overlapping the CNS tumor with BCOR internal tandem duplication (ITD) but lacking BCOR immunostaining and BCOR ITD. Instead, the tumor showed CREBBP::BCORL1 fusion and pathogenic mutations in BCOR and CREBBP, along with a DNA methylation profile matching the “CNS tumor with EP300:BCOR(L1) fusion” methylation class. Two CNS tumors with fusions between CREBBP, or its paralog EP300, and BCORL1, and approximately twenty CNS tumors with CREBBP/EP300::BCOR fusions have been reported to date. They exhibited similar ependymoma-like features or a microcystic pattern, along with focal or absent GFAP immunostaining, and shared the same DNA methylation profile. Given their morphological and epigenetic similarities, circumscribed CNS tumors with EP300/CREBBP::BCOR(L1) fusions and CNS tumors with BCOR ITD may represent variants of the same tumor type. The ependymoma-like aspect coupled with the lack of diffuse GFAP immunostaining and the presence of OLIG2 positivity are useful clues for recognizing these tumors in histopathological practice. The diagnosis should be confirmed after testing for BCOR(L1) gene fusions and BCOR ITD.

Clinical and morphological features of central nervous system tumor with <I>BCOR</I> internal tandem duplication

In 2022, central nervous system (CNS) tumor with BCOR internal tandem duplication (BCOR ITD) was included in the fifth edition of the World Health Organization Classification of Tumors of the CNS as part of the embryonal tumor group. The identification a distinct DNA methylation profile and the presence of a recurrent genetic aberration – BCOR ITD – made it possible to recognize these tumors as a separate entity. In most cases, these tumors occur in children under 5 years of age and are located in the hemispheres of the cerebellum or brain. Since CNS tumor with BCOR ITD is a rare and relatively new tumor entity, it is not yet widely known and can be misdiagnosed. The most common initial diagnosis in patients referred for a second opinion to our reference center is anaplastic/classical medulloblastoma. In this article, we aimed to summarize the characteristic clinical, morphological and immunophenotypic features of CNS tumors with BCOR ITD based on 8 clinical cases confirmed by molecular genetic testing. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology.

KDM2B-Rearranged Soft Tissue Sarcomas Expand the Concept of BCOR-Associated Sarcoma

Sarcomas with BCOR genetic alterations (BCOR-associated sarcomas) represent a recently recognized family of soft tissue and bone tumors characterized by BCOR fusion, BCOR internal tandem duplication, or YWHAE::NUTM2B fusion. Histologically, the tumors demonstrate oval to spindle cell proliferation in a variably vascular stroma and overexpression of BCOR and SATB2. Herein, we describe 3 soft tissue sarcomas with KDM2B fusions that phenotypically and epigenetically match BCOR-associated sarcomas. The cases included 1 infant, 1 adolescent, and 1 older patient. All tumors showed histologic findings indistinguishable from those of BCOR-associated sarcomas and were originally diagnosed as such based on the phenotype. However, none of the tumors had BCOR or YWHAE genetic alterations. Instead, targeted RNA sequencing identified in-frame KDM2B::NUTM2B, KDM2B::CREBBP, and KDM2B::DUX4 fusions. KDM2B fusions were validated using reverse-transcription PCR, Sanger sequencing, and in situ hybridization assays. Genome-wide DNA methylation analysis matched all 3 tumors with BCOR-associated sarcomas using the Deutsches Krebsforschungszentrum (DKFZ) classifier and t-distributed stochastic neighbor embedding analysis. One localized tumor showed a flat genome-wide copy number profile, and the patient remained disease-free after treatment. The other tumors showed multiple copy number alterations, including MDM2/CDK4 amplification and/or CDKN2A/B loss, and both tumors metastasized, leading to the patient’s death in one of the cases. When tested using KDM2B immunohistochemistry, all 3 KDM2B-rearranged sarcomas showed diffuse strong staining, and all 13 sarcomas with BCOR genetic alterations also demonstrated diffuse, strong, or weak staining. By contrast, among 72 mimicking tumors, only a subset of synovial sarcomas showed focal or diffuse weak KDM2B expression. In conclusion, our study suggests that KDM2B-rearranged soft tissue sarcomas belong to the BCOR-associated sarcoma family and expand its molecular spectrum. This may be related to the known molecular relationship between KDM2B and BCOR in the polycomb repressive complex 1.1. Immunohistochemical analysis of KDM2B is a potentially valuable diagnostic tool for BCOR-associated sarcomas, including those with KDM2B rearrangement.

3D genomics identify alternate mechanisms of homologous recombination deficiency in uterine sarcoma.

e17624 Background: Uterine sarcomas are rare tumors with diverse morphologic appearances, genetic underpinnings, and clinical behavior. Molecular characterization of these tumors by next generation sequencing (NGS) has improved disease classification and prognostication, but its impact on therapeutic selection remains limited. Analysis of 3D genomics datasets has recently been shown to detect large complex structural variants (SV) and chromatin architecture in normal and diseased human tissues. We applied genome-wide chromosome conformation capture (Hi-C) techniques in a variety of uterine sarcomas to identify novel therapeutic targets. Methods: Formalin-fixed paraffin-embedded tissue from 48 primary or recurrent uterine sarcomas were subjected to Hi-C to identify chromosomal rearrangements. The cohort included leiomyosarcoma (LMS, n = 7), adenosarcoma (AS, n = 7), endometrial stromal sarcoma (n = 7; high grade (HGESS), n = 4; low grade (LGESS), n = 3), sarcoma not otherwise specified (n = 17; high grade (HGNOS), n = 10, low grade (LGNOS), n = 7), undifferentiated uterine sarcoma (UUS, n = 6) and heterologous sarcoma (n = 2). Tumors were previously subjected to targeted DNA (n = 10) and/or RNA (n = 26) NGS with no therapeutic targets identified and only BCOR internal tandem duplication detected in a HGESS. Results: Hi-C detected SV in 73% of uterine sarcomas (n = 35/48), including those previously negative by targeted RNA NGS (n = 25/26). Homologous recombination repair (HRR) gene alterations were detected by Hi-C in 42% of tumors (n = 20/48), including AS (n = 5/7), HGESS (n = 2/4), LGESS (n = 1/3), LMS (n = 2/7), UUS (n = 2/6), heterologous sarcoma (n = 1/2), HGNOS (n = 3/10) and LGNOS (n = 1/7). SV included gene (gene to gene, n = 3; gene to non-coding DNA, n = 8) and proximity (n = 13) fusions disrupting HRR genes ( ATM, BRIP1, CDK12, CHEK1, CHEK2, NBN, PARP3, RAD51B, RAD51C, RAD51D) with enrichment for RAD51B fusions (50%, n = 10/20). Among all tumors with HRR gene alterations detected by Hi-C, only one uterine sarcoma harbored a missense ATM mutation by targeted DNA NGS (n = 1/5). Conclusions: Molecular characterization of uterine sarcomas by Hi-C may uncover HRR gene alterations not currently detectable by targeted DNA and RNA NGS. Homologous recombination deficiency (HRD) is not limited to LMS and may frequently result from SV, particularly between HRR gene to non-coding DNA in a variety of uterine sarcomas. These alternate mechanisms for HRD may serve as a potential therapeutic biomarker for HRD-targeted therapy in uterine sarcoma.

Clinical relevance of BCOR internal tandem duplication and TP53 aberration in clear cell sarcoma of the kidney.

Clear cell sarcoma of the kidney (CCSK) is the second most common pediatric renal malignancy, characterized by BCOR internal tandem duplication (ITD), YWHAE rearrangement, BCOR-CCNB3 fusion, and lack of other consistent structural alteration. We accidentally identified TP53 deletion in CCSK, which was often associated with adverse clinical outcomes. In this study, we assessed the incidence as well as the clinical relevance of these molecules in CCSK patients. BCOR ITD, YWHAE rearrangement, BCOR-CCNB3 fusion and TP53 status were examined by polymerase chain reaction, fluorescence in situ hybridization, or Sanger sequencing in a cohort of 39 patients with CCSK. Among them, 34 cases (87.18%) had BCOR ITD, 1 (2.56%) had YWHAE rearrangement, and 1 (2.56%) had BCOR-CCNB3 gene fusion. The remaining 3 (7.69%) harbored none of these aberrations. BCOR ITD, YWHAE rearrangement and BCOR-CCNB3 were mutually exclusive. Furthermore, 25.64% of the cohort acquired TP53 aberration (10/39, 3 with both copy number deletion and point mutation, 6 with deletion only, and 1 with mutation only), all of which were associated with BCOR ITD. Patients with or without BCOR ITD or TP53 aberration did not differ in demographic characteristics such as sex, onset age, or tumor stage at diagnosis. However, the overall survival rates and progression-free survival rates of BCOR ITD or TP53 deletion groups showed obvious downward trends, albeit not all reaching statistical significance. Patients with both BCOR ITD and TP53 deletion had the poorest prognosis.

CNS tumor with EP300::BCOR fusion: discussing its prevalence in adult population

The Central Nervous System (CNS) tumor with BCOR internal tandem duplication (ITD) has recently been added as a novel embryonal histomolecular tumor type to the 2021 World Health Organization (WHO) Classification of CNS Tumors. In addition, other CNS tumors harboring a BCOR/BCORL1 fusion, which are defined by a distinct DNA-methylation profile, have been recently identified in the literature but clinical, radiological and histopathological data remain scarce. Herein, we present two adult cases of CNS tumors with EP300::BCOR fusion. These two cases presented radiological, histopathological, and immunohistochemical homologies with CNS tumors having BCOR ITD in children. To compare these tumors with different BCOR alterations, we performed a literature review with a meta-analysis. CNS tumors with EP300::BCOR fusion seem to be distinct from their BCOR ITD counterparts in terms of age, location, progression-free survival, tumor growth pattern, and immunopositivity for the BCOR protein. CNS tumors from the EP300::BCOR fusion methylation class in adults may be added to the future WHO classification.

Molecular Signature of Biological Aggressiveness in Clear Cell Sarcoma of the Kidney (CCSK)

Clear cell sarcoma of the kidney (CCSK) is a rare pediatric renal tumor with a worse prognosis than Wilms' tumor. Although recently, BCOR internal tandem duplication (ITD) has been found as a driver mutation in more than 80% of cases, a deep molecular characterization of this tumor is still lacking, as well as its correlation with the clinical course. The aim of this study was to investigate the differential molecular signature between metastatic and localized BCOR-ITD-positive CCSK at diagnosis. Whole-exome sequencing (WES) and whole-transcriptome sequencing (WTS) were performed on six localized and three metastatic BCOR-ITD-positive CCSKs, confirming that this tumor carries a low mutational burden. No significant recurrences of somatic or germline mutations other than BCOR-ITD were identified among the evaluated samples. Supervised analysis of gene expression data showed enrichment of hundreds of genes, with a significant overrepresentation of the MAPK signaling pathway in metastatic cases (p < 0.0001). Within the molecular signature of metastatic CCSK, five genes were highly and significantly over-expressed: FGF3, VEGFA, SPP1, ADM, and JUND. The role of FGF3 in the acquisition of a more aggressive phenotype was investigated in a cell model system obtained by introducing the ITD into the last exon of BCOR by Crispr/Cas9 gene editing of the HEK-293 cell line. Treatment with FGF3 of BCOR-ITD HEK-293 cell line induced a significant increase in cell migration versus both untreated and scramble cell clone. The identification of over-expressed genes in metastatic CCSKs, with a particular focus on FGF3, could offer new prognostic and therapeutic targets in more aggressive cases.

Pediatric BCOR-Altered Tumors From Soft Tissue/Kidney Display Specific DNA Methylation Profiles

In the pediatric population, BCL6-correpresor gene (BCOR)–upregulated tumors include primitive myxoid mesenchymal tumors/undifferentiated sarcomas (PMMTI/UND), clear cell sarcomas of the kidney (CCSK), and high-grade neuroepithelial tumors (HG-NET). We investigated DNA methylation (DNAm) and copy number variation (CNV) profiling in these tumors (N = 34) using an Illumina EPIC BeadChip to better define the potential use of these tools to confirm diagnosis and predict outcomes. Twenty-seven tumors from 25 patients (age range, 0-10 years), showed molecular confirmation of genetic abnormalities as follows: BCOR internal tandem duplication in 14 PMMTI/UND, 8 CCSK, and 3 HG-NET and YWHAE fusions in 2 PMMTI/UND. The remaining 7 cases lacking informative molecular data were analyzed by immunophenotyping and were included in the study as a training cohort, clearly separated from the main study group. These were 4 PMMTI, 1 HG-NET, and 1 CCSK in which poor RNA preservation precluded the confirmation of BCOR rearrangements and 1 CCSK in which no rearrangements were found. DNAm data were compared with those of brain tumor and/or sarcoma classifier. Differentially methylated regions (DMRs) were analyzed in the 3 groups. Twenty-two cases of the 24 molecularly confirmed PMMTI/UND and CCSK and 3 of 6 of those with only immunophenotyping were classified within the methylation class “BCOR-altered sarcoma family” with optimal calibrated scores. PMMTI/UND and CCSK showed similar methylation profiles, whereas thousands of DMRs and significantly enriched pathways were evident between soft tissue/kidney tumors and HG-NET. The CNV analysis showed an overall flat profile in 19 of the 31 evaluable tumors (8/10 CCSK; 9/18 PMMTI/UND; 2/4 HG-NET). The most frequent CNVs were 1q gain and 9p and 10q loss. Follow-up time data were available for 20 patients: ≥2 CNV significantly correlated with a worse overall survival rate. In conclusion, soft tissue and kidney BCOR sarcomas matched with BCOR-altered sarcoma methylation class, whereas those from the brain matched with the central nervous system tumor classifier HG-NET BCOR, supporting the notion that DNAm profiling is an informative diagnostic tool. CNV alterations were associated with a more aggressive clinical behavior.

BCOR expression in paediatric pineoblastoma.

BCOR is expressed in a new brain tumour entity, i.e. 'CNS tumour with BCOR internal tandem duplication' (HGNET BCOR) but not in several other high grade paediatric brain tumours investigated. Immunohistochemical detection of BCOR expression may therefore serve as a potential diagnostic marker. Nevertheless, in rare paediatric glioma cases recurrent EP300-BCOR fusions were detected, which resulted in strong BCOR immunopositivity. We have therefore examined other, not analysed so far, types of central nervous system (CNS) tumours, pineoblastoma and germinoma, to assess a potential involvement of BCOR in these tumours. Levels of BCOR RNA expression were investigated by NanoString nCounter system analysis in a series of altogether 66 high grade paediatric tumours, including four pineoblastoma cases. Immunohistological detection of BCOR was performed in eight pineoblastoma, five germinoma and four atypical teratoid rhabdoid tumours (ATRTs), all located in the pineal region. We detected BCOR expression in all pineoblastomas, at the RNA and protein levels, but not in germinomas and ATRTs. Further analysis of pineoblastoma samples did not reveal the presence of either BCOR internal tandem duplication or BCOR fusion involvement. Positive immunohistological BCOR nuclear reaction in pineoblastoma may therefore differentiate this type of tumour from other high grade tumours located in the pineal region.

Expanding the spectrum of “mesenchymal” tumors of the central nervous system

In this review, we summarize the clinical, histopathological, and molecular features of central nervous system (CNS) tumors with BCOR internal tandem duplication, intracranial mesenchymal tumor with FET/CREB fusion, CNS CIC-rearranged sarcomas and primary intracranial sarcoma DICER1-mutant, now included in the 2021 WHO classification of CNS tumors. Possible relationships between tumors occurring in the CNS and their systemic counterparts are discussed.

CNS Tumor with BCOR Internal Tandem Duplication: The First Case in Southeast Asia

The central nervous system (CNS) tumor with BCOR internal tandem duplication (ITD) or high-grade neuroepithelial tumor with BCOR alterations (HGNET-BCOR) has been proposed as a new entity of CNS embryonal tumor. We report herein the first case of CNS tumor with BCOR ITD in Southeast Asia. The patient was a 2-year-old girl who presented with head tilt and ataxia. CNS showed a posterior fossa mass, 5.7x5.2x4.2 cm, with faint enhancement and internal hemorrhage. The lesion was removed, and the pathological examination revealed a hypercellular tumor comprising neoplastic cells with oval hyperchromatic nuclei. Perivascular pattern was occasionally observed. Pseudopalisading necrosis was seen but microvascular proliferation was absent. Although the clinical features of HGNET-BCOR are not distinctive and may overlap with other high-grade primary CNS tumors, awareness of the pathological features and the positive BCOR immunostain should lead to a final diagnosis with the identification of BCOR ITD using a molecular method.

Paediatric BCOR-associated sarcomas with a novel long spliced internal tandem duplication of BCOR exon 15.

Clear cell sarcoma of the kidney (CCSK) and primitive myxoid mesenchymal tumour of infancy (PMMTI) are paediatric sarcomas that most commonly harbour internal tandem duplications (ITDs) of exon 15 of the BCOR gene, in the range of 87–114 base pairs (bp). Some cases, instead, have BCOR‐CCNB3 or YWHAE‐NUTM2 gene fusions. About 10% of cases lack any of these genetic alterations when tested by standard methods. Two cases of CCSK and one PMMTI lacking the aforementioned mutations were analysed using Archer FusionPlex technology. Two related BCOR exon 15 RNA transcripts with ITDs of lengths 388 and 96 bp were detected in each case; only the 388 bp transcript was identified when genomic DNA was sequenced. In silico analysis of this transcript revealed acceptor and donor splice sites indicating that, at the RNA level, the 388‐bp transcript was likely spliced to form the 96‐bp transcript. The results were confirmed by Sanger sequencing using primers targeting the ITD breakpoint. This novel and unusually long ITD segment is difficult to identify by DNA sequencing using typical primer design strategies flanking entire duplicated segments because it exceeds the typical read lengths of most sequencing platforms as well as the usual fragment lengths obtained from formalin‐fixed paraffin‐embedded material. As diagnosis of CCSK and PMMTI may be challenging by morphology and immunohistochemistry alone, it is important to identify mutations in these cases. Knowledge of this novel BCOR ITD is important in relation to primer design for detection by sequencing, and using RNA versus DNA for sequencing.

CNS tumor with BCOR internal tandem duplication: Clinicopathologic, molecular characteristics and prognosis factors

The central nervous system tumor with BCOR internal tandem duplication (CNS tumor with BCOR ITD) is a recently identified rare tumor entity, the complete morphologic characteristic, genetic alteration, classification, clinical outcomes and optimal treatment for this tumor entity have not been fully clarified. Here, two new cases of CNS tumor with BCOR ITD were reported and the clinicopathologic, molecular characteristics, and prognosis were analyzed through reviewing of the reported literature. The histological features included a clear border with adjacent brain parenchyma, an extensively microcystic background, and the cells with round to oval nuclei containing delicate chromatin, ependymoma-like perivascular pseudorosettes, and palisading necrosis. Immunohistochemical features showed strong and diffuse positive expression for BCOR, scattered positive expression for OLIG2, and negative expression for GFAP, Syn, and EMA. PCR and direct DNA sequencing analysis identified exon 15 ITD of the BCOR gene in both cases. Interestingly, both cases revealed two duplication segments, which had not been reported in the literature. A review of the literature shows that CNS tumor with BCOR ITD has a poor prognosis with a median survival of 1.7 years; surgical gross total resection is a good prognostic factor. A combination of radiation treatment and chemotherapy, while trending towards significance, does not reach statistical significance. On the contrary, the OS is not associated with age, gender, and tumor location. In conclusion, CNS tumor with BCOR ITD is a rare tumor entity with characteristic morphologic and molecular features and a poor prognosis. The optimal treatment strategies need further studies.

ETMR-06. Molecular and clinical characteristics of CNS tumors withBCOR(L1) fusion/internal tandem duplication

Central nervous system (CNS) tumor with BCOR internal tandem duplication (BCOR-ITD) have recently been introduced in the 5th edition of the WHO classification of CNS tumors, however, their molecular makeup and clinical characteristics remain widely enigmatic. This is further complicated by the recent discovery of tumors characterized by gene fusions involving BCOR or its homologue BCORL1. We identified a cohort of 206 BCOR altered CNS tumors via DNA methylation profiling and conducted in-depth molecular and clinical characterization in an international effort. By performing t-SNE clustering analysis we found that BCOR-fusion tumors form a distinct cluster (n=61), adjacent to BCOR-ITD cases (n=145). The identified fusion partners of BCOR(L1) included EP300 (n=20), CREBBP (n=5), and NUTM2HP (n=1). Notably, three cases within the BCOR-ITD cluster harbored a c-terminal intragenic deletion within BCOR. With respect to clinical characteristics gender ratio was balanced in BCOR-fusion cases (m/f, 1.1), whereas predominance of male patients was observed in the BCOR-ITD group (m/f, 1.5). Moreover, age at diagnosis of BCOR-fusion patients was higher as compared to BCOR-ITD cases (15 vs 4.5 years). Interestingly, BCOR-fusion tumors were exclusively found in the supratentorial region being originally diagnosed as ependymomas or gliomas whereas BCOR-ITD emerged across the entire CNS with diverse original diagnoses. 8% of BCOR-ITD and none of BCOR-fusion cases were disseminated at diagnosis. In line with this observation, 40% of first relapses within the BCOR-ITD group were metastatic which was less frequent in BCOR-fusion tumors. Survival estimates demonstrated no differences, generally showing short median PFS (BCOR-fusion, 2 years, n=15; BCOR-ITD, 1.8 years, n=55) and intermediate OS rates (BCOR-fusion, 6.8 years, n=18; BCOR-ITD 6.3 years, n=60). Further molecular and clinical characterization is ongoing potentially revealing first therapeutic leads for these highly aggressive CNS tumor types.

ETMR-12. Novel cell models of CNS tumors with BCOR fusion or internal tandem duplication suggest FGFR and PDGFR as promising therapy targets

Central nervous system (CNS) tumors with BCOR internal tandem duplications (CNS-BCOR ITD) are aggressive malignancies recently included in the 2021 WHO Classification of CNS tumors. This entity is characterized by ITDs within the PUFD domain of BCOR, potentially interfering with protein-protein interactions and preventing non-canonical polycomb repressive complex 1.1 (ncPRC1.1) complex formation. Additionally, other BCOR alterations like frame shift mutations and gene fusions have been described. However, the underlying molecular mechanisms promoting tumor aggressiveness remain unknown. We established cell models from one patient harboring a BCOR frameshift mutation and another one with a concomitant BCORL1-fusion. Two additional models were derived from a patient with a CNS-BCOR ITD tumor. Multidrug screening uncovered high sensitivity against defined receptor tyrosine kinase (RTK) inhibitors (TKIs). In detail, ponatinib, nintedanib, and dovitinib reduced cell viability at half maximal inhibitory concentrations (IC50) in the low micro-molar range (<2.5 µM). Expression analyses of the respective TKI targets suggested fibroblast growth factor receptor 3 (FGFR3) and platelet derived growth factor receptor A (PDGFRA) as central players in this response. RTK inhibition resulted in strongly impaired downstream MAPK and Pi3K/AKT signaling. Vice versa, exposure to the RTK ligands bFGF and PDGFAA increased S6, Erk and Akt phosphorylation. Next, we treated two patients – one with a BCOR frame shift mutation/BCORL1-gene fusion and one with an ITD with nintedanib – within a multimodal treatment approach and achieving complete remission and disease stabilization, respectively. Ultimately, we analyzed respective RTK ligands in patient cerebral spinal fluid (CSF) and found FGF18 and PDGFA to correlate with tumor treatment response and progression. Summarizing, we uncover a central role of defined RTK signaling modules in the malignant phenotype of CNS-BCOR-ITD and tumors harboring BCOR alterations and elucidate their potential as therapeutic targets. Currently, we aim to dissect the interconnection between BCOR/BCORL1 alterations and RTK hyperactivation.

ETMR-11. Transcriptional changes upon knockdown of alteredBCOR/BCORL1 transcripts in preclinical models of CNS embryonal tumors with BCOR-related alterations

BCL-6 transcriptional corepressor (BCOR) is an epigenetic regulator that silences gene expression mainly via the polycomb repressive complex 1.1 (PRC1.1). BCOR genomic alterations are found in a variety of different tumors and recently central nervous system (CNS) tumors with BCOR internal tandem duplication (ITD) were classified as a distinct molecular subgroup. We established and characterized two cell models derived from BCOR altered CNS tumor patients. One model is characterized by a frameshift mutation in the BCOR gene resulting in the expression of a truncated protein lacking the C-terminal PUFD domain required for correct assembly of the PRC1.1. Additionally, this model harbors a translocation of the BCOR homologue BCORL1. The second model has a characteristic internal tandem duplication (ITD) within the BCOR gene. To study the effects of mutated BCOR/BCORL1 on gene expression, we performed siRNA mediated knockdown of altered BCOR/BCORL1 transcripts in both models and analyzed transcriptional changes by mRNA expression array. Differentially expressed genes in BCOR/BCORL1 knockdown versus wild type conditions were enriched for signaling pathways involved in cell cycle progression, cell growth, DNA replication and cancer. This suggests that the alterations in BCOR/BCORL1 might have pro-oncogenic effects and thereby contribute to the aggressive phenotype of this disease. Especially in the BCOR ITD model knockdown of BCOR led to transcriptional downregulation of genes associated with the development of brain tumors such as FGF18, PDGFA and PDGFRA. Our results indicate that specific BCOR/BCORL1 alterations might impair its endogenous function as transcriptional repressor and deregulate the expression of multiple PRC1.1 target genes. An in depth characterization of epigenetic and transcriptional changes in BCOR/BCORL1 altered CNS tumors could lead to the identification of critical downstream effectors and ultimately reveal new therapeutic vulnerabilities.

Endometrial Stromal Sarcomas With BCOR Internal Tandem Duplication and Variant BCOR/BCORL1 Rearrangements Resemble High-grade Endometrial Stromal Sarcomas With Recurrent CDK4 Pathway Alterations and MDM2 Amplifications.

The distinction between low-grade and high-grade endometrial stromal sarcomas (LGESS, HGESS) is increasingly defined by genetics. Recently, variant genomic alterations involving BCOR or BCORL1 have been reported in endometrial stromal sarcoma (ESS), although it remains unclear whether these justify a diagnosis of LGESS or HGESS. In this study, we describe clinicopathologic and molecular features of ESS with such alterations to help clarify their classification in the spectrum of ESS. We collected a cohort of 13 ESS harboring variant alteration involving BCOR (6 with internal tandem duplication, 1 with EP300::BCOR fusion, 1 with BCOR::LPP fusion) and BCORL1 ( 4 with JAZF1::BCORL1 fusion, 1 with EPC1::BCORL1 fusion). The median patient age at primary diagnosis was 51 years (range: 18 to 70 y). Median tumor size at primary diagnosis was 9.3 cm (range: 4.5 to 21 cm), and extrauterine disease spread (stage IIIB-C) was present in 27%. The tumors were composed of round to spindled cells with cellularity and cytologic atypia ranging from mild to marked and a median mitotic count of 18/10 HPFs (range: 2 to 85/10 HPFs). At least focally myopermeative growth was noted in 8/8 assessable cases. Of 12 patients with follow-up data (median: 25 mo), 4 patients died of disease and 3 were alive with recurrent disease. Unsupervised hierarchical clustering of DNA methylation data together with a large cohort of uterine mesenchymal tumors that included YWHAE::NUTM2 and Z C3H7B::BCOR HGESS and molecularly confirmed LGESS revealed a common methylation signature for all ESS with variant BCOR and BCORL1 alterations and HGESS with YWHAE::NUTM2 and ZC3H7B::BCOR gene fusion. Copy number analysis revealed amplifications of CDK4 and MDM2 , as well as homozygous deletions of CDKN2A/B and NF1 in a subset of tumors. Our results indicate that ESS with BCOR internal tandem duplication and variant BCOR and BCORL1 rearrangements clinically and molecularly resemble conventional HGESS.

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.