Genetic Alterations Research Articles

BCOR-ITD Rearranged Sarcoma in the Mandible of a 5-Year-Old Child: A Case Report Highlighting Diagnostic Distinction From Odontogenic Fibromyxoma.

The 5th edition of the World Health Organization (WHO) classification of soft tissue and bone has recognized three distinct groups among Ewing-like sarcomas, namely, CIC-rearranged sarcoma, sarcomas with BCOR genetic alterations, and round cell sarcomas with EWSR1:: non-ETS fusions. Sarcomas with BCOR genetic alterations are a distinct clinicopathological group of high-grade tumors, representing 5% of small round cell tumors. BCOR-ITD rearranged tumors commonly manifest as spindle cell sarcomas and many of them display low cellularity with monomorphous cell morphology and myxoid background resembling benign fibroblastic tumors. We present an intriguing case report of a 5-year-old boy diagnosed with a moderately cellular mandibular spindle cell tumor exhibiting BCOR-ITD rearrangement which was initially misdiagnosed as an odontogenic fibromyxoma. This case report illustrates the histological and immunophenotypic findings of BCOR-ITD rearranged sarcoma, requiring a comprehensive immunohistochemical panel and additional molecular tests for accurate diagnosis.

SURG-01. INTRAOPERATIVE FLUORESCENCE ACTIVITY OF 5-AMINOLEVULINIC ACID IN BRAIN METASTASIS OF LUNG CANCER IS ASSOCIATED WITH THE GENETIC ALTERATION OF CELL CYCLE REGULATION

Abstract With advancement of technology in brain tumor surgery, fluorescence image, such as 5-Aminolevulinic Acid (5-ALA), can help detect the residual tumor during surgery. Recently, genetic alteration of cancer is additionally considered as prognostic factor with being added into traditional prognostic system, such as Graded Prognostic Assessment (GPA) scoring system. The primary objective of this study was to investigate the association of certain genetic alterations and intraoperative fluorescent activity of 5-ALA in brain metastasis (BM) of lung adenocarcinoma. A retrospective cohort study was conducted among 72 patients who underwent surgical resection of BM of lung adenocarcinoma at our institute for five years. Cancer cell infiltration was estimated by the intraoperative fluorescent activity of 5-ALA, and genetic alterations were analyzed by next-generation sequencing (NGS). Total 72 patients were eligible for analysis. Strong fluorescence was obtained in 56 (77.8%) patient during surgery. The sensitivity and specificity for detecting cancer cell infiltration using 5-ALA were 87.5% and 96.4%, respectively. In the NGS data of BM, genetic alteration of cell cycle regulation, DNA repair, tumorigenesis, proliferation, epigenetic regulation, and cancer immunology were detected in 73.6%, 44.4%, 56.9%, 66.7%, 30.6%, and 2.8%, respectively. Genes associated with cell cycle regulation (p = 0.003) and cell proliferation (p = 0.044) were significantly associated with positive fluorescence activity of 5-ALA in the adjacent brain tissue. Genetic alterations in cell cycle regulation and cell proliferation were also associated with shorter recurrence-free survival (p = 0.013 and p = 0.042, respectively) and overall survival (p = 0.026 and p = 0.042, respectively) in the multivariate analysis. Additionally, age, KPS, status of NSCLC, RPA class, GPA score, modality of adjuvant treatment were associated with PFS and OS.The results suggest that genetic alterations in cell cycle regulation and cell proliferation are associated with positive fluorescence activity of 5-ALA in the adjacent infiltrative brain tissue and influence the clinical outcome of BM of lung adenocarcinoma.

EPCO-26. LONGITUDINAL SINGLE-CELL ANALYSES IDENTIFY DRIVERS OF GENETIC, EPIGENOMIC, AND CELLULAR EVOLUTION IN IDH-MUTANT GLIOMA

Abstract Despite standard treatment, IDH-mutant gliomas inevitably recur. Previous studies suggest that therapeutic resistance may result from a combination of intratumoral cellular heterogeneity, epigenetic evolution, and acquired genetic alterations. However, how these multilayered molecular features interact to influence the evolutionary paths of IDH-mutant gliomas remains incompletely understood. To chart this evolution, 75 glioma samples were longitudinally collected from 35 patients (n = 13 oligodendroglioma, n = 22 astrocytoma) and profiled using single nucleus RNA sequencing, ATAC sequencing, and bulk DNA sequencing. Analysis of 331,016 nuclei (snRNA) revealed eight tumor microenvironment cell types and five pan-IDH-mutant malignant cellular states. The malignant states were distributed along a cellular hierarchy of stem-like populations (neural progenitor cell-like, oligodendrocyte progenitor cell-like, and undifferentiated) and more differentiated populations (mesenchymal-like and astrocyte-like) with cycling cells enriched in the stem-like populations. Joint open chromatin accessibility data (snATAC) was available for a subset of malignant cells (71,088 nuclei) and state-specific differentially accessible peaks supported that these states are epigenetically encoded. Across both subtypes, higher tumor grade was associated with reduced malignant cell differentiation and increased cycling populations. Between the two time points, we identified that there was a longitudinal increase in the cycling, undifferentiated, and mesenchymal-like populations with a corresponding decrease in the astrocyte-like population. Longitudinal genetic analysis revealed that 19 of 35 tumors acquired at least one of the following genetic alterations: treatment-associated hypermutation, cell cycle alteration, or large changes in copy number alterations. Importantly, tumors that acquired these key genetic alterations demonstrated significant shifts towards reduced differentiation and increased cycling populations while those tumors that did not had more stable malignant profiles. Collectively, our results suggest a common cellular hierarchy across IDH-mutant gliomas with a shift towards reduced differentiation, increased cycling populations during disease progression that is driven by acquired genetic alterations.

CNSC-44. TARGETING OF THE YAP/TAZ-TEAD AXIS DIMINISHES VIABILITY AND DIFFERENTIATION POTENTIAL IN PEDIATRIC HIGH-GRADE GLIOMA

Abstract Pediatric high-grade gliomas (pHGGs) are invasive and fast-growing brain malignancies. They account for up to 40% of all brain tumor-related deaths in childhood. Genetic alterations that characterize these gliomas include H3 onco-histone mutations and genetic alterations in receptor-tyrosine kinases (RTKs), including amplification, mutation, and gene fusions in RTKs. However, direct targeting with tyrosine kinase inhibitors (TKIs) has shown at best partial responses in patients due to the molecular heterogeneity within these tumors and the lack of pharmaceutical penetration into the central nervous system (CNS). Thus, alternative therapeutic approaches to treat pHGGs are urgently needed. The YAP/TAZ transcription factors, which cooperate with TEAD co-factors to promote target gene expression, orchestrate stem-cell fate decisions and proliferation downstream of growth factor signaling pathways, such as RTKs. We observed that YAP expression is upregulated in RTK mutant and H3 mutant pHGGs. Depletion of YAP via RNAi methods decreased proliferation, inhibited pHGG stem cell self-renewal, and induced apoptosis of pHGG cells. Use of Verteporfin (VP), a FDA-approved YAP/TAZ small molecule inhibitor, mimicked the effects seen by YAP genetic depletion. Using an in vitro human cortical organoid-tumor model in which tumor cells are engrafted to human brain cortical organoids, we screened for responses in tumor cells and in non-tumor cells in the neural microenvironment upon VP treatment, using single-cell RNA-sequencing and immunofluorescence to visualize responses. These organoid models offer improved models for modeling how the neural CNS microenvironment shapes responses of tumor cells to YAP/TAZ inhibition and VP treatments. Results from our work showing that YAP/TAZ-TEAD inhibition is a promising therapeutic avenue for management of pHGGs will be presented. Further pre-clinical work will follow to correlate observation and understand the therapeutic efficacy of YAP/TAZ-TEAD inhibition for pHGG treatment.

RADT-23. MOLECULAR PROFILING OF GLIOMAS: INSIGHTS INTO RADIONECROSIS AND PSEUDOPROGRESSION THROUGH NEXT-GENERATION SEQUENCING

Abstract BACKGROUND Gliomas, the most common primary tumors of the CNS, presenting diagnostic and therapeutic challenges due to their heterogeneity. Next-generation sequencing (NGS) enhances diagnostic precision by identifying genetic alterations, guiding accurate diagnosis and personalized treatment strategies. Post-treatment complications such as radionecrosis and pseudoprogression present additional diagnostic difficulties, often mimicking tumor recurrence. This study aimed to evaluate the association between genetic alterations detected by NGS and treatment-related complications. METHODS This retrospective study analyzed data from electronic medical records at a Brazilian Radiation Therapy Department (2018-2023). The study included patients with gliomas who underwent surgical resection followed by adjuvant radiochemotherapy and subsequently developed complications such as radionecrosis and pseudoprogression. Genetic alterations detected by the HSL 500 (NGS) test were evaluated for their association with these complications using Fisher’s Exact Test. RESULTS Out of 192 patients treated for glioma, 46 (23.96%) experienced treatment-related complications, with 20 patients having available NGS test results. Among these, eight cases were identified as pseudoprogression, fifteen as radionecrosis, with three patients presenting both conditions. All 20 patients with NGS results were diagnosed with high-grade gliomas, eight of whom underwent reirradiation, resulting in two cases of radionecrosis post-reirradiation. Although no statistically significant association was found between genetic alterations and radionecrosis or pseudoprogression, specific mutations were prevalent in each group. In radionecrosis cases, the most common mutations were TERT promoter (c.1-124C>T) (n=9, p=0.613) and EGFR amplification (n=8, p=1.0). In pseudoprogression, frequent mutations included TERT promoter (c.1-124C>T) (n=6, p=0.642), EGFR (n=5, p=0.67), PTEN (n=4, p=1.0), NF1 (n=2, p=0.537), and P53 (n=2, p=0.197). CONCLUSION Molecular testing for glioma characterization is crucial for diagnosis and prognosis, aiding in understanding treatment-related complications. Strategies to avoid radionecrosis and pseudoprogression are vital to improve patient quality of life, especially in high-grade gliomas. Further studies are needed to confirm these trends and explore clinical relevance of mutations.

PATH-35. INTEGRATED GENOMIC ANALYSES OF IMMUNODEFICIENCY-ASSOCIATED EPSTEIN-BARR VIRUS- (EBV) POSITIVE PRIMARY CNS LYMPHOMAS

Abstract Immunodeficiency-associated primary CNS lymphoma (PCNSL) represents a distinct clinicopathological entity, which is typically Epstein-Barr virus positive (EBV+) and carries an inferior prognosis. Genetic alterations that characterize EBV related CNS lymphomagenesis remain unclear precluding molecular classification and targeted therapies. In this study, a comprehensive genetic analysis of 22 EBV+ PCNSL, integrated clinical and pathological information with exome and RNA sequencing (RNASeq) data. Deconvolution of bulk RNA sequencing data was performed using CIBERSORTx to characterize the tumor microenvironment. EBV+ PCNSL with germline controls carried a median of 55 protein-coding single nucleotide variants (SNVs; range 24-217) and 2 insertions/deletions (range 0-22). Genetic landscape was largely shaped by aberrant somatic hypermutation with a median of 41.01% (range 31.79-53.49%) of SNVs mapping to its target motifs. Tumors lacked established SNVs (MYD88, CD79B, PIM1) and copy number variants (CDKN2A, HLA loss) driving EBV negative PCNSL. Instead, EBV+ PCNSL were characterized by SOCS1 mutations (26%), predicted to disinhibit JAK/STAT signaling, and mutually exclusive gain-of-function NOTCH pathway SNVs (26%). Copy number gains were enriched on 11q23.3, a locus directly targeted for chromosomal aberrations by EBV, that includes SIK3 known to protect from cytotoxic T-cell responses. Losses covered 5q31.2 (STING), critical for sensing viral DNA, and 17q11 (NF1). Unsupervised clustering of RNASeq data revealed two distinct transcriptional groups, that shared strong expression of CD70 and IL1R2, previously linked to tolerogenic tumor microenvironments. Correspondingly, deconvolution of bulk RNASeq data revealed elevated M2-macrophage, T-regulatory cell, mast cell and monocyte fractions in EBV+ PCNSL. In addition to novel insights into the pathobiology of EBV+ PCNSL, the data provide the rationale for the exploration of targeted therapies including JAK-, NOTCH- and CD70-directed approaches.

EPCO-18. DECIPHERING SPATIAL DIVERSITY OF GLIOBLASTOMA GENETIC ARCHITECTURE

Abstract Glioblastoma (GBM) is hallmarked by profound inter- and intratumoral heterogeneity. Current research has mapped key genetic drivers to distinct transcriptional profiles within GBM. Nevertheless, the intricate interactions between genetic alterations and spatial-transcriptional heterogeneity remain elusive. We utilized nestin-Tva C57BL/6 mice to induce de novo GBM via oncogene overexpression alongside shRNA-mediated silencing or deletion of tumor suppressors. We modeled critical driver mutations: PDGFB, NF1, and EGFR. Employing 10X Genomics platform for single-cell RNA sequencing, combined with spatially resolved transcriptomics and proteomics (Nanostring GeoMx), we integrated data using both horizontal (mutual nearest neighbors) and vertical (weighted nearest neighbors) approaches, analyzed through the SPATA2 software. Advanced computational techniques, including graph neural networks and single-cell deconvolution, were leveraged to unravel the genotypic and spatial variability within tumors and their microenvironments. Our findings uncover persistent cellular heterogeneity across diverse genetic contexts. However, specific genetic mutations precipitated distinct dominant transcriptional states: NF1 and EGFR mutations drove mesenchymal and astrocytic-like states, respectively, whereas PDGFB fostered a neural progenitor/cell-like state. These transcriptional shifts were closely linked to unique microenvironmental modifications, such as elevated neuronal signaling in PDGFB-driven GBMs and significant immune infiltration in NF1 and EGFR models. Through weighted co-expression network analysis (WGCNA) and gene set enrichment analysis (GSEA) paired with spatial-niche deconvolution, we identified a mesenchymal transition leading to an immunosuppressive microenvironment in NF1-loss driven GBMs and increased lymphocyte presence around blood vessels in EGFR-amplified GBMs. These patterns were consistent with human spatially resolved transcriptomic data, emphasizing the genetic complexity and heterogeneity of human GBMs. Our genetically engineered mouse models serve as a robust platform for dissecting GBM with defined genetic mutations. Leveraging sophisticated analytical techniques and computational modeling, this study underscores the profound interplay between genetic drivers and the tumor microenvironment, paving the way for targeted therapeutic strategies and precision medicine in GBM.

Exploring the Incidence of Testicular Neoplasms in the Transgender Population: A Case Series.

The use of hormonal therapy and gender-affirming surgery in the transgender community has been rising during the last several years. Although it is generally safe, hormonal therapy's link to testicular cancer remains uncertain. To review the incidence of testicular cancer in specimens from gender-affirming orchiectomies at our institution and evaluate the tumors for histologic and genetic alterations. Pathology reports for gender-affirming orchiectomies (January 1, 2018, to August 1, 2023) were reviewed for testicular neoplasms, with additional analysis for chromosome 12 abnormalities. Incidence and chromosome variations were compared with those in the general population. Among 458 cases during 5.5 years, 5 germ cell neoplasms in 4 patients emerged. Our institution's annual incidence rate (159 per 100 000) is 26.5 times higher than the National Cancer Institute's previous report (6.0 per 100 000). Although they were morphologically no different from germ cell neoplasms in the general population, fluorescence in situ hybridization tests showed no i(12p) in 4 of 5 neoplasms (80%) in our cohort. The cause behind this rise in incidence remains uncertain but may be due to long term pretreatment with hormones or blockers. The lower isochromosome 12p frequency suggests an alternative mechanism driving tumor development, which requires more detailed molecular studies.

CTNI-48. A PHASE 1 STUDY OF ERAS-801, A POTENT, SELECTIVE, AND CENTRAL NERVOUS SYSTEM (CNS)-PENETRANT EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) INHIBITOR, FOR PATIENTS WITH RECURRENT GLIOBLASTOMA MULTIFORME (GBM)

Abstract EGFR is among the most common targets for genetic alterations in GBM. Changes in EGFR gene amplification and the extracellular domain result in receptor overexpression or constitutive activation, and enhanced oncogenic signaling. ERAS-801 is a potent, selective, and highly CNS-penetrant EGFR inhibitor being developed for the treatment of GBM, with a focus on tumors harboring EGFR genetic alterations. THUNDERBBOLT-1 is the first-in-human phase 1 dose escalation and expansion trial evaluating ERAS-801 monotherapy for patients with recurrent GBM. ERAS-801 is administered orally once daily for continuous 28-day cycles. As of April 10, 2024, a total of 52 patients were treated across 7 dose cohorts: 20 mg (n=3), 40 mg (n=5), 80 mg (n=3), 160 mg (n=6), 240 mg (n=25), 280 mg (n=4), or 320 mg (n=6). Key patient characteristics were median age of 60 years, 67% male, mostly one prior therapy, and 81% tumors with alterations. The MTD was 240 mg. Dose-limiting toxicities were observed at 320 mg (n=2, Grade 2 QT prolongation, Grade 3 rash and Grade 3 dermatitis acneiform), 280 mg (n=3, Grade 3 QT prolongation), and 240 mg (n=1, Grade 3 QT prolongation) doses. The most common treatment related adverse events (TRAEs), in >10% of patients, were dermatitis acneiform, diarrhea, and nausea. Electrocardiogram QT prolongation occurred in 11.5% of patients and was not associated with clinical findings. ERAS-801 showed rapid absorption; peak plasma concentration was generally achieved within 6 hours post dose. PK exposure increased in a dose-dependent manner over the doses evaluated. Across all doses there was a confirmed partial response (using mRANO) in 1 patient and 6 patients achieved a PFS of 6 months. ERAS-801 shows well behaved PK with substantial CNS penetration, preliminary safety, and tolerability in patients with recurrent GBM. The TRAEs were reversible, manageable, and consistent with known toxicities of EGFR inhibitors.

DDDR-35. THERAPEUTIC STRATEGIES FOR DIFFUSE MIDLINE GLIOMA: INSIGHTS FROM HIGH-THROUGHPUT COMBINATION DRUG AND RADIATION SENSITIZATION SCREENING

Abstract Diffuse midline gliomas (DMGs) are the most aggressive pediatric high-grade gliomas. Over 85% DMGs harboring the H3K27M mutation in histone genes H3F3A (H3.3) and H3C2 (H3.1). H3K27M mutation disrupts epigenetic regulation by inhibiting PRC2, causing loss of H3K27 trimethylation and oncogenic gene expression. Presently, there are no effective therapies for DMG, and 95% of the patients die within 1.5 years of diagnosis. Thus, there is an urgent need to identify novel therapeutic strategies targeting H3K27M oncohistone. To assess the H3K27M mutation’s vulnerability, we used paired isogenic H3K27M-mutated and CRISPR-corrected H3M27K (H3-WT) primary DMG cell lines derived from thalamus (BT-245) and pons (DIPGXIIIP). We performed comprehensive single-agent high-throughput screens (HTS) with 1,520 FDA-approved drugs and further assessed drug combinations of ONC201 with the 86 most promising drugs from the HTS. A third combination experiment evaluated radio-sensitizing potential, where most effective top-six drugs (A-F) were tested in combination with radiation therapy across an expanded panel of patient-derived DMG cell cultures. To test drug efficacy in vivo, we developed a genetically engineered isogenic mouse model (GEMM) of DMGs using intrauterine electroporation (IUE) with DNp53/PDGFRA/H3.3K27M (PPK) and DNp53/PDGFRA/H3.3WT (PPW) genetic alterations. Our initial single-agent HTS screening identified 86 drugs with significantly higher cytotoxicity, specifically against H3K27M-mutated DMG cell lines. Combination HTS of the top six drugs revealed that drug E displayed significantly synergistic cytotoxicity with ONC201. We also evaluated drugs A-E in combination with radiation in both human and mouse H3K27M-mutated DMG cell lines. Drugs D and E showed selective radiosensitivity in both human and mouse H3K27M-mutated DMG cell lines. Preliminary findings also suggested drug D acts as an anti-invasive agent. Our findings identify novel drugs via HTS that enhance ONC201 efficacy and act as radiation sensitizers in DMGs. Future studies will test these compounds with ONC201 and radiation in our in vivo IUE-mediated H3K27M mouse model.

New therapies for hormone-positive HER2-negative metastatic breast cancer with AKT signaling alterations: the Expert Panel Decision

On May 20, 2024, the Expert Panel discussed key issues of the diagnosis of alterations of the AKT signaling pathway, the effectiveness and safety of targeted therapy for hormone-positive (HR+) HER2-negative metastatic breast cancer (BC) concerning the emergence of a new selective AKT inhibitor capivasertib. The results of the CAPItello-291 study are presented. The experts concluded that a new effective treatment line has become available for patients with hormone-resistant advanced hormone-positive HER2-negative BC with genetic alterations in the AKT signaling pathway and relapse on previous adjuvant endocrine therapy with aromatase inhibitors or within the first year after the therapy or with progression during previous therapy with aromatase inhibitors for metastatic BC. Modern genetic testing methods for the alterations in the AKT signaling pathway can confirm the presence of this key biological target in the tumor for tailoring effective targeted therapy.

NIMG-01. RADIOMIC DATA ANALYSIS WITH ENSEMBLE MACHINE LEARNING TECHNIQUES PREDICT GRADE, HISTOPATHOLOGIC SUBTYPE, AND 1P/19Q CO-DELETION STATUS IN PATIENTS WITH LOW-GRADE GLIOMAS

Abstract Low-grade gliomas (LGGs) are a diverse group of primary brain tumors characterized by their variable prognosis and treatment response. Traditionally, the management of LGGs has relied on histopathological analysis, which sometimes offers limited insight into tumor behavior and treatment outcomes. Recent advances in radiomics have enabled the extraction of detailed quantitative features from routine imaging, providing a non-invasive method to assess tumor characteristics. These radiomic features can capture underlying tumor heterogeneity more comprehensively than conventional methods. Integrating radiomic data with ensemble machine learning techniques offers a promising approach to enhance the prediction of tumor grade, histopathologic subtype, and genetic alterations such as 1p/19q co-deletion status in LGGs, potentially leading to more tailored therapeutic strategies. We combined radiomic-genomic data from 159 magnetic resonance imaging (MRI) scans from the updated LGG-1p19qDeletion dataset (Erickson 2020) and 114 MRI scans from the ReMIND dataset (Juvekar 2024). Expert radiologists performed segmentations. A total of 2446 extracted radiomic features were initially extracted. Feature selection was performed using wrapper- and filter-based techniques with cross-validation. Prediction models were built using machine learning techniques, including random forest and extreme gradient boosting. Using Random Forest, we achieved high accuracy in predicting histopathologic subtype (88.14%, AUC 96.68%) with precision of 87.86% and Dice score of 87.88%. For grade prediction (accuracy 76.19%, AUC 91.84%), precision was 73.91%, and the Dice score was 77.27%. For 1p/19q co-deletion status (accuracy 75.61%, AUC 82.74%), precision was 77.78%, and the Dice score was 73.68%. Our study establishes a robust radiomic workflow to predict tumor grade, histopathologic subtype, and 1p/19q co-deletion status in low-grade gliomas, enhancing diagnostic accuracy and treatment personalization. This methodology can be applied clinically to improve the stratification of treatment plans for low-grade gliomas, potentially leading to effective, personalized therapy options. Using multicentric data potentially improved external validity, something which past research lacked. Future research should focus on validating and deploying this model using observational multicentric study designs.

TMET-17. LIPID METABOLIC HETEROGENEITY OF GLIOMA CELLULAR STATES REVEALS ENVIRONMENTAL DEPENDENCIES

Abstract Malignant growth and survival of cancer is supported through reprogrammed lipid metabolism. In gliomas, genetic alterations can drive lipid metabolic dysregulation revealing therapeutic opportunities to exploit lipid metabolic vulnerabilities within genetically defined subsets of glioma tumors. However, it remains unclear whether inter- and intra-tumoral transcriptomic heterogeneity in gliomas relates to distinct lipid programs and potential dependencies. Here we set out to characterize pan-glioma lipid metabolic heterogeneity through genomic, transcriptomic, and lipidomic profiling of a large and diverse cohort of patient tumor samples. We define key axes of lipid metabolic variability across gliomas and identify relationships between lipid metabolic gene expression programs and glioma lipidomic profiles. Intriguingly, intersection of lipid gene expression signatures with single cell RNA sequencing revealed patterns of lipid metabolic heterogeneity that distinguish neurodevelopmental cellular states of gliomas, mirroring patterns of lipid metabolic diversity across cell types within the normal brain. Consequently, tumors with opposing cellular state enrichment have distinct lipid metabolism and environmental dependencies. Tumors enriched for Radial Glia-like states have high capacity for de novo fatty acid synthesis while tumors enriched for oligodendrocyte progenitor-like states are dependent on exogenous sources of lipids for survival and growth. These findings connect inter- and intra-tumoral heterogeneity of cellular states to variability in lipid metabolic reprogramming to reveal future avenues for precision medicine.

EPCO-21. ELUCIDATING THE FUNCTIONAL RELEVANCE OF ATRX-DEFICIENCY IN H3.3-G34-MUTANT DIFFUSE HEMISPHERIC GLIOMA

Abstract Diffuse hemispheric gliomas (DHGs) account for 30% of aggressive cerebral tumors in children and young adults, exhibiting abysmal outcomes. Co-existing recurrent mutations in H3.3 histones at residue G34 (H3.3-G34R/V mutation) and concurrent inactivating mutations in TP53 and α-thalassemia/intellectual disability-X-linked gene (ATRX) implicate a coordinated molecular effort to drive oncogenesis. ATRX, a core component of the SWItch/Sucrose Non-Fermentable chromatin remodeling complex, regulates the incorporation of histone H3.3 into chromatin sites across the genome to maintain epigenome integrity. Yet, it is unclear how ATRX loss combines with H3.3-G34R/V mutations to influence cellular phenotype and dysregulate neuro-developmental programs. We propose that ATRX deficiency coordinates with key oncogenic partners by inducing oncogenic transcriptional programs and neuro-developmental pathways in DHGs. To investigate this, we employed CRISPR-editing to model H3.3-G34R followed by ATRX knockdown (ATRX-KD) in a TP53 mutant (TP53-mut) human-induced pluripotent stem cell (h-iPSC) line, developing an isogenic system to map contributions to epigenetic dysfunction made by specific molecular alterations. Upon differentiation of our h-iPSCs to neural stem cells (NSCs), we found unique transcriptional profiles associated with each genetic alteration, with upregulation of forebrain progenitor genes driven by ATRX-loss. Hierarchal clustering of normalized transcript counts revealed gene signatures from each model driving unique functional pathways, with the ATRX-KD and the triple mutant (TP53-mut, ATRX-KD, H3.3-G34R) signatures enriched for neural development pathways. Additionally, both signatures were associated with published transcriptional profiles of G34-mutant DHGs, emphasizing the disease relevance of our models. Further, time-course analysis using cerebral organoids uncovered distinct morphological changes with each alteration, with the triple mutant exhibiting impaired differentiation at later time points. These latter findings suggest that ATRX deficiency transcriptionally influences early neuronal differentiation, effects further refined by H3.3-G34 mutation. To conclude, our innovative models demonstrate the involvement of ATRX in neuro-developmental and differentiation phenotypes, illuminating underlying molecular features of aggressive DHGs.

EPCO-27. CHROMATIN REMODELING REPROGRAMS THE TRANSCRIPTION NETWORK IN GLIOBLASTOMA

Abstract Fundamentally, cancer arises from genetic and epigenetic alterations that interplay to reprogram gene expression networks, leading to unrestrained proliferation. Glioblastoma (GBM) is the most prevalent and aggressive primary brain cancer, with a median survival of approximately one year and a 5-year survival rate of just 5%. The standard care for GBM patients has remained unchanged for decades, underscoring the need for a better understanding of GBM biology to develop more effective therapies. The relatively low genetic mutation burden in GBM highlights the significant role of epigenetic mechanisms in its pathogenesis. Our recent work shows that chromatin remodeling plays pivotal roles in GBM pathogenesis. The BRD8-driven EP400 chromatin remodeling complex maintains GBM by crippling p53-mediated tumor suppression in an unprecedented way through hijacking the histone variant H2AZ at p53 target loci, enforcing a repressive chromatin state that prevents p53-mediated transactivation. Targeting BRD8 in TP53WT GBM, which makes up ~71% of all GBM cases, enhances chromatin accessibility by evicting H2AZ. This restores p53-mediated transactivation of its targets, reestablishes cell cycle arrest, inhibits gliomagenesis, and prolongs survival in xenograft models of TP53WT GBM. Conversely, the NuRD chromatin remodeling complex driven by CHD5 governs the MYC-mediated oncogenic network to prevent gliomagenesis. Mechanistically, CHD5 forms a CHD3-containing but CHD4-independent NuRD complex that directly binds to the MYC promoter and super enhancers. Chd5 loss triggers the remodeling of chromatin, enhances chromatin accessibility at Myc loci, augments Myc expression, significantly impairs NSCs differentiation while promotes proliferation, transformation, and gliomagenesis in vivo. Furthermore, reactivating CHD5 in human GBM xenograft models significantly extends survival. Thus, our findings reveal previously unappreciated epigenetic mechanisms by which GBM cells reprogram both tumor-suppressive and oncogenic transcription networks to facilitate their outgrowth. This sheds new light on our understanding of GBM malignancy and provides promising therapeutic opportunities for treating patients with this devastating malignancy.

BIOM-46. REAL-WORLD EXPERIENCE WITH CIRCULATING TUMOR DNA IN CEREBROSPINAL FLUID IN PATIENTS WITH CENTRAL NERVOUS SYSTEM TUMORS

Abstract Next generation sequencing (NGS) of circulating tumor DNA (ctDNA) from cerebrospinal fluid (CSF) offers a minimally invasive approach to identify somatic alterations for individuals with central nervous system (CNS) cancers. Here, we review our ‘real world’ experience using a clinically validated, FDA-authorized platform, MSK-IMPACTTM, to comprehensively profile ctDNA from CSF samples with and without clinically documented CNS involvement by cancer (1007 samples, 711 patients). The patient cohort included patients with over 90 distinct tumor types of primary CNS and metastatic origin, with lung cancer (n = 188), breast cancer (n = 150), and glioma (n = 148) representing the most common broad categories. We identified genetic alterations (ctDNA-positive) in 489/922 (53.0%) CSF samples from patients with clinically documented CNS cancer. Over half of these ctDNA-positive samples (248, 50.7%) possessed a biomarker that was predictive of response to an FDA-approved drug (OncoKB level 1). Furthermore, the distribution of clinically actionable somatic alterations was consistent with tumor-type specific alterations across the AACR GENIE cohort. Of patients without clinical documentation of CNS cancer, none of these 85 CSF samples were ctDNA-positive (specificity = 100%). Successive CSF sequencing over time identified clonal evolution and the emergence of resistance mechanisms to therapies. ctDNA positivity corresponded with shortened overall survival after CSF collection (HR: 3.23, 95% CI: 2.58-4.05, P < 0.001). NGS of CSF cfDNA can detect clinically actionable somatic alterations in a large subset of CNS cancer patients and allows tracking of tumor evolution. Detection of somatic alterations in CSF may be a useful prognostic biomarker for survival in cancer patients.

BIOM-43. SOMATICTP53, PIK3R1, NF1 ORTERT MUTATIONS ARE ASSOCIATED WITH INFERIOR CLINICAL OUTCOME IN H3K27M-ALTERED DIFFUSE MIDLINE GLIOMA: AN INTERNATIONAL, MULTI-INSTITUTIONAL STUDY

Abstract BACKGROUND H3K27-altered diffuse midline gliomas (DMGs) have poor prognosis with no standard of care therapy beyond radiation (RT). While RT prolongs survival, not all patients respond. Molecular biomarkers can be a strategy to predict RT response and thus patient outcomes. METHODS We performed a retrospective analysis of patients with biopsy-proven H3K27M-altered DMG between 2013-2023 from six US medical centers and the DMG Center in Zurich. Patients with tumor genomic sequencing and completion of RT were eligible. Genomic alterations were evaluated for somatic mutations, fusions, and chromosomal instability. Cox proportional hazard models were used to evaluate associations between genomic alterations and survival. Multivariate analysis included all significant variables at P <0.1 in initial analysis, including age at diagnosis, tumor location, TP53 and PIK3R1 alterations. RESULTS 297 patients (135 female; median age 8.3 years; range 0.2-71.4 years) were included. Median progression-free survival (PFS) and overall survival (OS) was 7.6 months (95%CI: 6.9-8.6) and 14.0 months (95%CI: 12.9-15.7). In univariate analysis, TP53 or PIK3R1 status correlated with shorter OS (TP53-mutant 12.5 vs. TP53-wildtype 17.5 months; HR=1.5; 95%CI: (1.1-2.0), P= 0.005); (PIK3R1-mutant 12.6 vs. 14.5 months; HR=1.9, 95%CI: (1.1-3.4), P = 0.03). Multivariate analysis corroborated TP53-mutant status with poorer OS (HR=1.5; 95%CI: (1.2-2.0), P= 0.003). Subgroup univariate analysis of pontine DMG patients showed poorer OS with TP53, NF1, or TERT mutations (TP53-mutant 11.9 vs. 15.2 months; HR= 1.6; 95%CI: (1.1-2.3), P= 0.02); (NF1-mutant 8.4 vs. 13.6 months; HR= 2.3; 95%CI: (1.1-5.0), P=0.03); (TERT 8.5 vs. 13.6 months; HR= 2.5; 95%CI: (1.1-5.7), P= 0.03). Subsequent multivariate analysis corroborated the association of TERT mutations with poorer OS in pontine DMG (HR=2.5; 95%CI:1.05, 5.9, P= 0.03). CONCLUSIONS This is the largest cohort of H3K27M-altered DMG patients with volumetric imaging analysis ongoing. Taken together, our data suggests these genetic alterations can risk-stratify patients for RT and future investigations of radiosensitizers targeting these alterations may improve survival.

INNV-10. ULTRA-RAPID INTRAOPERATIVE GLIOMA TUMOR CELL QUANTIFICATION

Abstract The diagnosis and treatment of brain tumors often depends on molecular-genetic data. To date, however, there has been no intraoperative method to rapidly and iteratively utilize tumor-specific genetic alterations to guide brain tumor surgical resections, complicating intraoperative diagnosis and precluding measurement of tumor cell burdens at surgical margins. To address this gap, we developed Ultra-Rapid droplet digital PCR (UR-ddPCR), which can be completed in 15 minutes from tissue to result with an accuracy and sensitivity comparable to standard ddPCR. We demonstrate an UR-ddPCR assay for the IDH1 R132H clonal mutation that is present in many low-grade gliomas. We illustrate the clinical feasibility of UR-ddPCR by performing it intraoperatively for 49 samples from 13 glioma cases, achieving intraoperative quantification in an average of 14 minutes and 54 seconds spanning a wide dynamic range from 0.1% to 94% tumor cell percentages. We further combine UR-ddPCR measurements with UR-stimulated Raman histology intraoperatively to estimate tumor cell densities in addition to tumor cell percentages, across a wide range from < 1 to 1332 cells/mm2. We anticipate that UR-ddPCR, along with future refinements in assay instrumentation, will enable novel point-of-care diagnostics and the development of molecularly-guided brain tumor surgeries that improve clinical outcomes.

CNSC-41. DABRAFENIB AND TRAMETINIB AS A PROMISING TREATMENT OPTION FOR PEDIATRIC POPULATION WITH LOW-GRADE GLIOMAS THAT HAVE BRAF V600E MUTATION:A BREAKTHROUGH IN THE FIELD OF NEURO- ONCOLOGY

Abstract Two-thirds of all pediatric malignant central nervous tumors, including high-grade (glioblastoma, anaplastic astrocytoma) and low-grade (ganglioglioma, pilocytic astrocytoma) carcinomas, are gliomas. Low-grade glioma(LGG) exhibits genetic alterations caused by the BRAF kinase mutation, such as replacing glutamic acid (E) in place of valine (V) at the 600 positions, known as the V600E point mutation. Pediatric low-grade gliomas (PLGGs) also comprise around one-third of juvenile brain tumors and are the most frequent central nervous system tumors. Patients were randomized 2:1 to dabrafenib plus trametinib (D+T) or carboplatin plus vincristine (C+V). Where possible, complete surgical removal is the mainstay of treatment for progressing or symptomatic PLGG.Radiation therapy has historically been used to treat PLGG in both up-front and salvage scenarios. To delay or avoid the necessity for radiation therapy in young children with advancing or incompletely resected PLGG, chemotherapy was created in 1980. On March 16, 2023, the Food and Drug Administration approved the use of trametinib (Mekinist, Novartis) with dabrafenib (Tafinlar, Novartis) in pediatric patients suffering from low-grade glioma (LGG) who require systemic therapy and are at least one year old. Nevertheless, early-stage clinical trials have produced encouraging results that may revolutionize the treatment of LGG in the near future.

MET amplification correlates with poor prognosis and immunotherapy response as a subtype of melanoma: a multicenter retrospective study.

Mesenchymal epithelial transition factor (MET) variant is an independent prognostic factor for worse prognosis in patients with lung cancer or gastroesophageal adenocarcinoma. MET gene variants can be regarded as a subtype of melanoma but there is a lack of studies regarding the frequency of MET genetic alterations and the efficacy of immunotherapy in melanoma patients. The purpose of this study is to explore potential therapeutic strategies for melanoma subtypes with MET alterations. A total of 1751 malignant melanomas were analyzed to illustrate the landscape of MET mutations. We collected 55 melanoma cases from multicenter for a retrospective cohort from 2010 to 2023. We analyzed the impact of MET amplification on the efficacy of immunotherapy in the retrospective cohort after propensity score matching (PSM) and a pancancer cohort. CIBERSORT was used to evaluate the immune infiltration. There were no instances of MET 14 exon skipping, and only instances of MET amplification were found in the 1751 melanomas and our retrospective cohort. Cox proportional hazards model analysis showed that MET amplification (P = 0.006) was significantly associated with poorer overall survival (OS) in patients who received immunotherapy as the first-line treatment. Compared with patients with MET amplification, patients in the negative control (NC) group had a significantly better OS (P = 0.022) after PSM. Analysis of 1661 pancancer cases with the MSK-IMPACT assay showed that patients receiving immunotherapy in the MET amplification group had a trend toward worse OS than those without MET amplification (P = 0.025). This database analysis showed that the main type of MET mutation is amplification in malignant melanoma. MET-amplified solid tumors might be considered for targeted therapy, as MET amplification can be regarded as a risk factor affecting the prognosis of patients with tumors treated with immunotherapy.