Next-generation Sequencing Research Articles

PATH-06. TRANSLATION INTO CLINICAL PRACTICE OF THE G1-G7 MOLECULAR SUBGROUP CLASSIFICATION OF GLIOBLASTOMA

Abstract Glioblastoma is the most frequent and malignant primary brain neoplasm. Glioblastoma growth is promoted by alterations in mediators from five major pathways: MAPK and PI3K canonical growth pathways, telomere elongation/TERT, cell cycle G1-phase and the p53 pathway. In a recent breakthrough study on a prospective Discovery cohort, I proposed the first all-inclusive molecular classification of glioblastoma into seven subgroups, G1-G7, based on MAPK pathway activation. New data from a subsequent, prospective Validation cohort validated the G1-G7 molecular subgroup classification, showing significant demographic and molecular differences between subgroups. Beside the initial histologic diagnosis, the G1-G7 classification requires next generation sequencing (NGS) of formalin-fixed paraffin-embeded (FFPE) samples and genomic-transcriptomic correlations between DNA mutations, copy number variations (CNVs) and RNA expression. Thus, in the Combined cohort containing tumors from over 220 glioblastoma patients, aproximately 75% of cases fell into three major subgroups, G1/EGFR, G3/NF1 and G7/Other, whereas the rest of 25% cases made up the four minor subgroups, G2/FGFR3, G4/RAF, G5/PDGFRA and G6/Multi-RTK. The name of the mutated most upstream, non-redundant, MAPK pathway effector corresponds to each subgroup, except for the G7/Other subgroup in which MAPK activation is minimal and PI3K pathway activation is dominant. The effectors of the other four pathways may be redundant; however, genomic-transcriptomic correlations showed subgroup specificity, with opposing trends for the G1/EGFR and G7/Other molecular subgroups. This first, all-inclusive, molecular subgroup classification of glioblastoma in a large prospective patient cohort allowed integrated pathway analysis, risk stratification and demographic comparison between Caucasian/White and African-American/Black patients. Most importantly, its implementation into clinical practice opened the avenue to controlled therapeutic approaches.

The Expression of HPV-16 E5 Oncoprotein Impacts the Transcript Profiles of FGFR2 and EMT-Related Genes in Preneoplastic Anal Epithelium Lesions

Anal Squamous Cell Carcinoma (SCCA) is a rare Human Papillomavirus type 16 (HPV16)-associated carcinoma whose pathogenesis is still poorly understood. Recent studies based on biopsy and Next Generation Sequencing (NGS) approaches have linked the viral episomal status to aggressive SCCA phenotypes, suggesting a potential role of the 16E5 oncoprotein in tumor development. Our previous findings indicated that 16E5 induces Fibroblast Growth Factor Receptor 2 (FGFR2) isoform switching, aberrant mesenchymal FGFR2c expression, Epithelial Mesenchymal Transition (EMT), and cell invasion in various in vitro human keratinocyte models, as well as in the in vivo context of cervical Low-grade Squamous Intraepithelial Lesions (LSILs). To further explore the role of 16E5 in epithelial carcinogenesis, this study aims to investigate the molecular profile in HPV-related anal lesions. The results showed a significant positive correlation between 16E5 and FGFR2c, as well as 16E5 or FGFR2c and key EMT-related transcription factors, particularly in the group of HPV16 positive anal samples not containing without high grade lesions. Additionally, by coupling the molecular analysis with an interactome investigation, we hypothesized a potential functional interplay between the Ca2+ channel Transient Receptor Potential Ankyrin 1 (TRPA1) and FGFR2c, mediated by 16E5 during the establishment of the oncogenic signaling. These findings will help to elucidate the actual relevance of 16E5 in the early progression of anal lesions and contribute to determine its potential as target for future preventive approaches for HPV16-positive SCCA.

PATH-41. PRIMARY BRAIN SARCOMA WITH DROP METASTASES AND LEPTOMENINGEAL DISEASE

Abstract INTRODUCTION Primary brain sarcoma is extremely rare and has a very poor prognosis. Sarcomas are a highly diverse group, with over 100 different subtypes identified in the recent WHO classification. Case: In this case, we describe a 51-year-old man diagnosed with primary right frontal poorly differentiated sarcoma. He experienced cognitive changes for weeks. Brain MRI revealed a lobulated mass with peripheral enhancement measuring 67 mm by 61 mm by 52 mm. He underwent gross total resection, and pathology indicated high-grade poorly differentiated sarcomatous lesions. Further analysis at Memorial Sloan Kettering and NIH failed to match any DNA methylation class. Next generation sequence (NGS) testing revealed mutation of NF2, CDKN2A, COL3A1, CD33, and PIK3CA genes, increased PD-L1 mRNA, and PD-L1 expression, greater than 50%. The conclusive diagnosis was a PD-L1 high poorly differentiated sarcoma, not otherwise specified. Since no primary site was found elsewhere, planned to treat it as a primary brain sarcoma. Patient underwent focal radiation therapy with a plan to start immunotherapy combination of anti-CTLA4 and anti-PD1 therapy. In addition, an anti-VEGF therapy with pazopanib because of the efficacy of pazopanib in the setting of soft tissue sarcomas. The patient completed IMRT on 07/13/2023 and presented with a new onset of left leg weakness and urinary incontinence on 07/25/2024, further neuraxis imaging with MRI showed local and distant intracranial recurrence with drop metastasis at C1, C5-C6, and S2. The patient was started on IMRT to address drop metastasis, however further clinical decline led to treatment discontinuation. DISCUSSION There is no consensus on treatment guidelines on primary brain sarcomas given that they are exceedingly rare. DNA methylation and NGS provided very valuable information. We were unable to find any reported cases of primary brain sarcoma with drop metastases and leptomeningeal disease in the literature.

Rapid diagnosis of culture-negative Klebsiella pneumoniae liver abscesses by next-generation sequencing: A case series

Diagnosis of Klebsiella pneumoniae (Kp) pyogenic liver abscesses is usually achieved by imaging and isolation of the bacterium. However, when blood and other cultures were negative, laboratory diagnosis of Kp liver abscess may be challenging. Herein we report two patients with culture-negative Kp liver abscess with atypical presentations diagnosed by metagenomic next-generation sequencing (mNGS). For the first non-diabetic patient, computed tomography examination of the abdomen showed multiple round low-density foci with untidy margin in the liver, which mimicked hydatid cysts; while the second patient presented with acute cholecystitis/cholangitis. mNGS analysis of the blood sample from the first patient revealed 144 sequence reads of Kp; and that of the second revealed 153 sequence reads of Kp as well as other latent or non-pathogenic microorganisms. Both patients responded promptly to antibiotics treatment. mNGS is a useful tool for laboratory diagnosis of culture-negative Kp liver abscess.

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.

PATH-59. HISTOLOGIC CORRELATES FOR MOLECULAR FEATURES IN MENINGIOMAS

Abstract Histologic findings are the primary determinant of meningioma prognosis and post-operative management. However, the association of specific pathologic features with molecular alterations has not yet been comprehensively investigated. Here we utilized a large dataset of well-characterized meningiomas to identify correlates between clinically relevant histological and molecular features. Detailed histologic information was collected for 809 resected meningiomas, including cases with DNA methylation (n=380), copy number array (n=252), next-generation sequencing (n=52), RNA risk score (n=394), and/or TERT promoter profiling (n=238). Analyses were conducted on prognostic pathologic features, including WHO Grade, Ki-67, mitotic index (MI), brain invasion, and atypical histologic findings (necrosis, hypercellularity, prominent nucleoli, sheeting, and small cells). Correlation with molecular variables was performed using Fisher’s Exact Tests, including DNA methylation subgroup, RNA risk score, CNV profiles, and TERT promoter mutations. All statistics were corrected for multiple hypothesis testing. Nearly 65% of meningiomas exhibited at least one atypical histologic feature. Among said features, Ki-67/MI, WHO Grade, sheeting, and necrosis were associated with recurrent lesions and known clinical features of aggressive meningiomas. Several histologic features, including hypercellularity, elevated Ki-67/mitotic index, and WHO grade, also correlated with DNA methylation and RNA risk groups. Several chromosomal arm events were related to specific pathologic features (prominent nucleoli, Ki-67, MI, and grade), including chromosomes 1, 4, 10, 14, and 18. Our analysis highlights the continued value in screening for histologic markers of aggressive behavior in meningiomas, as they correlate with the presence of high-risk molecular features and may serve as proxy markers when advanced molecular testing is not available.

AI-enabled pipeline for virus detection, validation, and SNP discovery from next-generation sequencing data

Background and AimsThe rapid and accurate detection of viruses and the discovery of single nucleotide polymorphisms (SNPs) are critical for disease management and understanding viral evolution. This study presents a pipeline for virus detection, validation, and SNP discovery from next-generation sequencing (NGS) data. The pipeline processes raw sequencing data to identify viral sequences with high accuracy and sensitivity by integrating state-of-the-art bioinformatics tools with artificial intelligence.MethodsBefore aligning the reads to the reference genomes, quality control measures, and adapter trimming are performed to ensure the integrity of the data. Unmapped reads are subjected to de novo assembly to reveal novel viral sequences and genetic elements.ResultsThe effectiveness of the pipeline is demonstrated by the identification of virus sequences, illustrating its potential for detecting known and emerging pathogens. SNP discovery is performed using a custom Python script that compares the entire population of sequenced viral reads to a reference genome. This approach provides a comprehensive overview of viral genetic diversity and identifies dominant variants and a spectrum of genetic variations.ConclusionThe robustness of the pipeline is confirmed by the recovery of complete viral sequences, which improves our understanding of viral genomics. This research aims to develop an auto-bioinformatics pipeline for novel viral sequence discovery, in vitro validation, and SNPs using the Python (AI) language to understand viral evolution. This study highlights the synergy between traditional bioinformatics techniques and modern approaches, providing a robust tool for analyzing viral genomes and contributing to the broader field of viral genomics.

Rhegmatogenous Retinal Detachment Secondary to Type I Stickler Syndrome: Diagnosis, Treatment and Long-Term Outcomes

Objective: This study aimed to clarify the genetic diagnosis of rhegmatogenous retinal detachment (RRD) secondary to type I Stickler syndrome (STL1) and evaluate the anatomical and functional outcomes of surgical treatment. Methods: This retrospective study included 11 patients with RRD secondary to STL1. Familial and sporadic cases of STL1 were diagnosed at the Eye & ENT Hospital, Fudan University, between 2017 and 2023. To clarify the genetic diagnosis, next-generation sequencing was performed in suspected STL1 cases. Further, standard ocular examinations and surgical treatment were performed. Results: Nine variants of COL2A1, including four novel mutations (c.394G>T, c.2977G>T, c.3003+2dup, and c.3853G>C), were screened and identified. The pathogenicity of all variants was conclusively demonstrated. Among patients who underwent vitrectomy, the mean age at RRD was 11.5 years, and the mean follow-up was 32.9 months. The average number of surgical procedures required during the follow-up was two; 90.9% of eyes achieved final attachment, and best corrected visual acuity (BCVA) significantly improved in 81.8% of the eyes, with a middle postoperative logMAR BCVA of 0.52 compared with the preoperative value (p = 0.0148). High intraocular pressure (81.8%) and cataract (72.7%) were the most common complications. Conclusions: Our study expands the spectrum of COL2A1 mutations and provides a novel diagnostic strategy for STL1. By combining clinical manifestations with genetic testing, STL1 could be accurately diagnosed. With proper surgical treatment and long-term follow-up, the prognosis of RRD in patients with STL1 could be improved.

BIOM-56. HEAT SHOCK PROTEINS PREDICT CLINICAL RESPONSE AND POTENTIATE LOCAL IMMUNOGENICITY AFTER LITT FOR GLIOBLASTOMA

Abstract Glioblastoma (GBM) remains one of the deadliest cancers despite advances in therapies. Laser interstitial thermal therapy (LITT) is a minimally invasive technique used to ablate intra-axial brain tumors. Non-ablative LITT-induced hyperthermia (33-43˚C) increases intra-tumoral mutational burden and neoantigen production, promoting neoantigen presentation via heat shock proteins (HSPs). HSPs are protein-folding chaperones that present novel intracellular antigens for immune recognition. We investigated HSPs as predictors of local immune response and clinical outcomes of GBM patients undergoing LITT. We conducted immune deconvolution of bulk RNA-sequencing data from The Cancer Genome Atlas to determine the correlation between 97 unique HSPs and tumor infiltration of CD8+, CD4+, B, macrophage, neutrophil, and dendritic cells. We evaluated the association between HSP expression and overall survival (OS). Pre-LITT biopsies from 9 patients (responders and non-responders) were analyzed via immunohistochemistry (IHC) to identify spatial HSP expression. Next-generation sequencing was utilized to validate findings and identify other predictive biomarkers. We identified two HSPs for further investigation: SACS and HSPA5. SACS was associated with improved survival based on TCGA data (14.9 vs. 11 months, HR:1.3, p<0.0001). HSPA5 was associated with worse outcomes (12.1 vs. 15.0 months, HR:0.73, p<0.0001). Immune deconvolution showed HSPA5 positively correlated with dendritic cell infiltration (r=0.50, p=6.1E-27) and SACS with CD8+ infiltration (r=0.31, p=6.8E-11). IHC of 9 patients demonstrated high SACS expression was associated with improved OS (p<0.05) after LITT. Multiplex IHC illustrated that immune cell infiltration was associated with therapeutic response post-LITT. Our ongoing immunohistochemical and genomic analyses aim to clarify the role of HSPs in enhancing immunogenicity in GBM. Further investigation into SACS and immune cell infiltration may reveal how hyperthermia via LITT improves response through neoantigen presentation.

BIOM-70. CSF TUMOR CELL (CSF-TC) DETECTION, QUANTIFICATION AND BIOMARKER ASSESSMENT HELPS IN CLINICAL MANAGEMENT OF BREAST CANCER AND NON-SMALL CELL LUNG CANCER PATIENTS HAVING LEPTOMENINGEAL DISEASE (FORESEE STUDY, NCT05414123)

Abstract Leptomeningeal metastases (LM) or the presence of cancer cells in the cerebrospinal fluid (CSF)/pia remains a challenging disease to diagnosis and treat. Current standard of care methods to diagnose or assess treatment response of LM (Clinical Evaluation, MRI and Cytology) have limited sensitivity and specificity and are poorly quantifiable. This creates challenges for physicians to manage LM or determine the best course of treatment. The FORESEE Study was a multi-center, prospective clinical trial evaluating a novel diagnostic platform, CNSide, that aimed to overcome these challenges. The FORESEE study enrolled patients with breast or non-Small Cell Lung Cancer (NSCLC) with suspicion of or confirmed LM. CNSide can detect and quantify tumor cells in the CSF from patients with breast cancer or NSCLC having a suspicious or confirmed LM. This platform also is able to identify actionable mutations in the CSF via Fluorescent In Situ Hybridization (FISH) and next-generation sequencing. The primary end point of the FORESEE study was clinical utility and the impact of CNSide on physician treatment decisions. Secondary endpoints included evaluating the clinical performance of CNSide vs. cytology in tumor cell detection (sensitivity, specificity, PPV and NPV). The study enrolled 40 patients (22 breast cancer and 18 NSCLC). Each patient underwent standard of care diagnostic evaluations at baseline and at three follow up timepoints through their treatment. CNSide aided clinical decision making in 93% (50/54) of the clinical decisions. At baseline 39 patients were assessed and confirmed a positive LM diagnosis in 15% (6/39), a negative LM diagnosis in 8% (3/39), progression in 15% (6/39), resolution in 15% (6/39) and no progression, or resolution in 36% (14/39) of the patients. CNSide informed the specific drug selected for treatment in 26% (10/39) patients at baseline and demonstrated clinical high utility. The FORESEE data is undergoing ongoing analysis; updated analysis will be presented at the meeting.

BIOM-53. LOW-PASS WHOLE GENOME SEQUENCING OF CEREBROSPINAL FLUID IDENTIFIES TUMOR AGNOSTIC TP53 ABERRATIONS IN LEPTOMENINGEAL METASTATIC DISEASE

Abstract Leptomeningeal metastatic disease (LMD) is the spread of cancer cells into the cerebrospinal fluid (CSF)-filled spaces surrounding the central nervous system (CNS). Once LMD occurs, patients endure devastating neurologic symptoms and survive for only a few weeks to months. The clinical diagnosis of LMD has risen as patient survival from non-CNS metastatic disease improves. Unfortunately, detection of LMD even in symptomatic patients is <50% using the current gold standard – CSF cytology. Therefore, a strong clinical need exists for the sensitive and early detection of LMD. Here, we sought to develop a minimally invasive liquid biopsy approach to diagnose LMD using next-generation sequencing. Because copy number variations (CNVs) are common in cancer and increase after chemoradiation, we hypothesized that CNV detection in CSF may offer a means to detect LMD since patients have already undergone treatment for the underlying disease. Specifically, we sought to develop low-pass whole genome sequencing (lpWGS) of cell-free DNA in CSF to detect CNVs associated with LMD. A novel lpWGS algorithm was developed using FASTQ files trimmed to 30 million total paired reads. The genome was partitioned into one million continuous base pair segments excluding problematic areas yielding 2,445 regions across 22 autosomes. Control samples were used to model the read depth for each region to establish the euploid state. Read depth across the genome for all samples was 0.6X. CSF samples from lung cancer, breast cancer, and melanoma patients with LMD demonstrated prominent evidence of monosomies and trisomies throughout the genome. Lung cancer exhibited frequent amplifications (>4-fold). Notably, we observed 17p monosomy in CSF regardless of cancer type which indicates TP53 haploinsufficiency may support LMD progression, a conjecture supported by a codeletion of TP53 in one patient. lpWGS detects LMD in solid tumor cancers and may also identify novel mechanisms for LMD progression.

DISP-17. OUTCOMES, CLINICAL AND MOLECULAR CHARACTERISTICS OF HISPANIC PATIENTS WITH IDH WILDTYPE GLIOBLASTOMA

Abstract BACKGROUND Knowledge about outcomes in Hispanic patients with gliomas is scant and mainly derived from tumor registries. IDH wild-type glioblastomas (GBM) are the most common and aggressive malignant primary brain tumor. Hispanics account for 19% of US population, reaching 29% in Florida and 65% in Miami-Dade County. As a reference center for GBM serving the community of South Florida we provide care for many Hispanic patients. Herein, we aim to describe the clinical and molecular features, including RNA seq analysis of Hispanic vs non-Hispanic patients with IDH wild-type GBM. METHODS Retrospective study of adult patients with diagnosis of IDH Wild-type Glioblastoma, who had NGS testing, treated at University of Miami. RESULTS 179 patients were included, 63 (35%) were Hispanic. 41 (65%) of Hispanic patients were males as compared to 65 (56%) of the non-Hispanics. Median age at diagnosis was 54 years (45-67) for the Hispanic vs 62 (54-72) years for non-Hispanic, p=.002. With a median follow up of 17 months (CI 95% 14-19), median overall survival for Hispanic patients was 22 months (18-25) and 23 months (18-28) for non-Hispanics, p=.99. 42 (23%) patients were enrolled in clinical trials, of whom 21 (50%) were Hispanic, meaning 33% of Hispanic patients enrolled. The molecular characteristics from these patients by next-generation sequencing, including RNA seq, are being analyzed. CONCLUSIONS In our series of IDH wild-type Glioblastomas, Hispanic patients were significantly younger at diagnosis compared to non-Hispanic patients, but there were no differences in overall survival. 33% of Hispanic patients were enrolled in clinical trials. As the latest growing ethnic group in the USA, major efforts have to be made to better understand the disease characteristics and outcomes of this particular population. To our knowledge this will be the first report of the molecular characteristics by NGS from Hispanic patients with GBM.

QLTI-09. CLINICAL TRIALS IN NEURO-ONCOLOGY: DESIGN, LOGISTICS, CHALLENGES, AND PRACTICAL CONSIDERATIONS

Abstract Timely, secure and reliable transportation, precise handling and storage of biological specimens are critical for the success of clinical research. Research in neuro-oncology often involves complex assays such as next-generation sequencing where the integrityof brain tumor specimens is paramount. These challenges are pronounced in most Lower Middle-Income Countries (LMIC), due to climatic factors and infrastructure limitations, necessitating stringent measures to preserve sample quality and their genetic data integrity for downstream analysis. In this study, we conducted extensive cohort analysis on 80 tissues randomly picked samples of the 3600 samples collected across India to assess the impact of collection protocols, transportation logistics, and long-term storage on the genetic material of tissues. The quality and quantity of the isolated nucleic acids was determined by BioAnalyzer and Qubit parameters. Tissue samples were collected and stored at -80°C in RNAstable® within 4-12 hrs of collection. The DNA and RNA obtained from tissue samples stored for over one year was found to be of good quality with corresponding DNA Integrity Number (DIN) and RNA Integrity Number (RIN)between 7-9. Also, optimum quantity of DNA and RNA was obtained for whole exome and transcriptome sequencing. Upon sequencing, these samples showed good read coverage of 100X or more. These findings provide critical insights into the measures necessary for optimizing sample preservation and transportation to ensure accurate research outcomes. The results indicate that appropriate sample collection, transportation, and long-term storage of bio-specimens can be used effectively for future research. Additionally, the outcome underscores the potential for long-term studies in neuro-oncology using archived biological samples without compromising data integrity.

Personalized MRD Assessment in Peri-surgical ctDNA for Prognostic Prediction in Hepatocellular Carcinoma

Abstract Objective: Detecting residual disease is a critical clinical requirement in the peri-surgical management of patients with resectable hepatocellular carcinoma (HCC). Previous studies focused on specific genomic regions exhibiting limited sensitivity and failed to meet the minimal residual disease (MRD) testing threshold. We introduce a next-generation sequencing (NGS) based assay, informed by baseline samples, facilitating MRD detection in hepatectomized HCC patients and offering prognostic predictions. Experimental Design: This study involved 88 HCC patients who underwent surgical resections January 2016 to May 2016 in Zhongshan Hospital Fudan University. Tumor and normal tissue samples were collected during surgery, while plasma samples were obtained both before surgery and up to seven days post-surgery. Using an NGS-based personalized circulating tumor DNA assay, we analyzed MRD in both pre-surgical and post-surgical blood samples and the correlation with prognosis. Results: With a median follow-up period of 80.7 months, our findings demonstrated significant correlations between pre-surgical ctDNA tumor fractions, post-surgical plasma MRD status, and both recurrence-free survival (RFS) and overall survival (OS). Post-surgical MRD status emerged as the most significant risk factor for cancer recurrence (HR=2.17, 95% CI: 1.09-4.30, p=0.027) compared to other clinical characteristics in multivariate Cox regression analysis. Notably, MRD status showed potential as a prognostic indicator among clinically low-recurrent-risk patients, such as those with BCLC stage 0-A, CNLC stage I-II. Conclusion: Evaluating personalized MRD provided crucial prognostic insights into RFS and OS. It efficiently identified patients at high risk of recurrence, even among those initially perceived as low-risk cases.

BIOM-07. SURVIVAL-ASSOCIATED GENETIC VARIATIONS IN TAIWANESE GLIOBLASTOMA MULTIFORME PATIENTS - A RETROSPECTIVE NGS STUDY

Abstract This retrospective study, conducted at Taichung Veterans General Hospital in Taiwan, employed Next-Generation Sequencing (NGS) technology to analyze genetic variations associated with survival in patients with glioblastoma multiforme (GBM). The study encompassed 30 newly diagnosed GBM patients, divided into two groups based on survival duration: long-term (over two years) and short-term (under two years). The findings are outlined below: Key Mutations: Significant mutations included CHEK2, IDH1, and TP53, along with variations in the TERT promoter and TP53 RNA splicing sites. Correlation Between Mutations and Survival: Contrary to Western data, TP53 mutations were more prevalent in the long-term survival group, suggesting that regional genetic differences may influence GBM prognosis. Therapeutic Effectiveness: The application of bevacizumab was significantly associated with improved survival rates, indicating its potential efficacy in treatment. The study highlights the critical role of personalized medicine in the treatment of GBM in Taiwan and points to the necessity of further research into the genetic characteristics and treatment responses of GBM patients in Taiwan and potentially other non-Western regions. These insights underline the importance of continuing to explore effective personalized treatment strategies and enhancing our understanding of genetic determinants in diverse populations. Moreover, the prevalence of certain mutations in Taiwanese patients, which differ from those commonly observed in Western populations, prompts a reevaluation of global treatment frameworks and suggests the potential for region-specific therapeutic protocols. The implication of these findings extends beyond immediate clinical applications, providing a foundation for future clinical trials and research into genetic markers that could dictate treatment choices. In conclusion, this study not only underscores the importance of individualized treatment plans based on genetic profiling but also highlights the variations in genetic mutations across different populations, which could have profound implications for the management of GBM worldwide. Further investigation into these disparities and their implications on treatment efficacy is essential for advancing GBM treatment and improving patient outcomes globally.

PATH-45. TREATMENT OF FGFR1 – ALTERED WHO GRADE 1 GLIONEURONAL TUMORS WITH FGFR INHIBITOR ERDAFITINIB

Abstract INTRODUCTION Low-grade mitogen-activated protein kinase (MAPK) pathway-altered glioneuronal tumors carry distinct molecular alterations, which may represent therapeutic opportunities to avoid or delay toxic treatments like radiation. Erdafitinib is a pan-FGFR tyrosine kinase inhibitor, regulatorily approved in recurrent urothelial carcinoma, and is under clinical investigation for recurrent FGFR-altered gliomas (NCT05859334). METHODS We report two patients with FGFR1-altered rosette-forming glioneuronal tumors (RGNT) treated with erdafitinib. RESULTS Patient 1 is a 32-year-old woman who presented with obstructive hydrocephalus and a non-enhancing pineal tumor. She underwent a subtotal resection, with histopathology initially consistent with a low grade dysembryoplastic neuroepithelial tumor. She was under observant management for 7 years, and developed worsening symptoms with slow non-enhancing growth, although stable per RANO low grade criteria. The initial tissue was sent for next-generation sequencing (NGS) and DNA methylation profiling, revealing an FGFR1 pathogenic variant with DNA methylation profiling matching with RGNT. Patient began Erdafitinib 8 mg daily, and had stable disease per RANO at 6 months. She developed CTCAE grade 1 toxicities of subretinal fluid, fatigue, dry skin, and paronychia. Patient 2 is a 34-year-old man diagnosed 1 year prior with a tectal tumor, who underwent biopsy due to non-measurable/ non-enhancing progression. Histopathology was consistent with RGNT. NGS revealed FGFR1 mutation, PIK3CA mutation, and NF1 pathogenic variant, described in the literature as characteristic of RGNT. There was insufficient tissue for DNA methylation profiling. Patient started erdafitinib 8 mg daily and remains clinically stable for 1 month. He developed asymptomatic hyperphosphatemia (CTCAE grade 1). Both patients have tolerated treatment without interruptions or dose reductions. CONCLUSION Erdafitinib is a well-tolerated and potentially efficacious treatment of FGFR1- altered RGNT, and may avoid the need for radiation. Diagnosis of MAPK- altered low-grade glioneuronal tumors, specifically RGNT, is increasingly reliant on molecular data, and can significantly impact treatment options.

NIMG-60. SUBTYPING GLIOBLASTOMA FROM JOINT CLUSTERING OF IMAGING AND GENOMIC DATA

Abstract PURPOSE To comprehensively unravel the heterogeneity of glioblastoma, we developed a subtyping method using integrated imaging and genomic data. METHODS We assembled a retrospective cohort of 571 IDH-wildtype glioblastoma patients, obtaining pre-operative multi-parametric MRI (T1, T1-GD, T2, T2-FLAIR, DSC, DTI) scans (available for 462 patients) and targeted next-generation sequencing (NGS) data (available for 355 patients). We extracted 971 radiomic features from these MRI scans, selecting 12 significant dimensions through L21-norm minimization guided by 13 key driver genes from the five most frequently altered pathways in glioblastoma compared to healthy controls (RB1, P53, MAPK, PI3K, and RTK). Subtypes were identified using a joint clustering approach that integrated radiomics and genomics. RESULTS Our method identified three glioblastoma subtypes with distinct risk levels: high-risk (subtype 1), medium-risk (subtype 2), and low-risk (subtype 3), each characterized by their differential overall survival outcomes (Kaplan-Meier analysis, p < 0.05). The intensities of axial diffusivity in enhancing tumor regions and radial diffusivity in non-enhancing cores increased across three subtypes. All subtypes shared a common tendency for more frequent mutation co-occurrence pattern in [TP53,RB1] but exhibited unique molecular characteristics. For example, subtype 1 displayed a relatively high frequency of co-occurring mutations in [TP53,KDR] and [NOTCH2,MDM4]; subtype 2 had six co-occurring pairs [TP53,KDR], [PTPN11,NF1], [RB1,FUBP1], [PTEN,CIC], [PTEN,KRAS], and [NOTCH2,CDKN2A]; subtype 3 showed six co-occurring pairs [PTPN11,NF1], [PDGFRA,NF1], [NTRK1,NOTCH2], [MET,ATRX], [KRAS,KDR], and [FGFR2,DNMT3A] plus four significant patterns of mutual exclusivity in [TP53,PTPN11], [TP53,EGFR], [EGFR,RB1], and [EGFR,NF1]. CONCLUSION Our study discovered distinct glioblastoma subtypes, revealing complex patterns and structures across multiple data modalities without relying on prior clinical assumptions. This is promising to help enhance the precision of diagnosis and treatment of glioblastoma.

BIOM-63. LONG-TERM SURVIVAL AND MOLECULAR CHARACTERIZATION OF IDH-WILDTYPE GLIOBLASTOMA IN AN ASIAN COHORT

Abstract BACKGROUND Glioblastoma (GB), the most aggressive form of brain tumor, presents a significant clinical challenge with a devastating prognosis. Globally, the median overall survival (OS) for GB is typically 12-15 months, largely due to its inherent resistance to chemotherapy and radiotherapy. However, some patients exhibit prolonged survival, and their specific demographic and genomic characteristics remain poorly defined. Understanding the clinical profile of these exceptional responders holds great promise for informing improved management strategies for GB. METHODS In a retrospective two-center study conducted in Hong Kong (IRB: UW 21-189), we recruited 80 patients diagnosed with IDH wildtype glioblastoma. All patients underwent next-generation sequencing as part of their treatment. Long-term survivor (LTS) was defined as >=36 months from diagnosis, while medium-term survivor (MTS) was defined as >=18 months, and <18 months as short-term survivors (STS). When appropriate, descriptive statistics were used to report frequency with percentage or median with range. Fisher’s exact test was employed to analyze categorical variables, while continuous variables were assessed using the Kruskal-Wallis test. All statistical analyses were conducted using R (version 4.2.3). Result: Among the cohort of 80 recruited patients, the median survival was 18.8 months (95% CI 17.2-24.8). Notably, 21% (n=17) were LTS, 31% (n=25) were MTS, and 48% (n=38) were STS. A statistically significant variation (p < 0.05) was evident across three groups: gender distribution, the number of resections performed, the proportion of patients receiving genomic-guided treatment, and their participation in clinical trials. However, no significant difference was observed in MGMT promoter status and ECOG score. CONCLUSION Identifying these clinical factors enhances the clinical management of GB. The implications of these findings underscore the potential importance of incorporating genomic-guided treatment in both local and global clinical practices in GB.

CSIG-16. A CX43-WNK1-C-MYC SIGNALING AXIS DRIVES GLIOBLASTOMA CANCER STEM CELL SURVIVAL

Abstract Tumors represent a highly dynamic system that relies on coordinated signaling to drive growth and adapt to selective pressures, including those induced by anti-cancer therapies. Given the need for tumors to engage in rapid and coordinated cell-cell communication, mechanisms such as gap junction intracellular communication (GJIC) should be essential. However, the connexin proteins that make up gap junctions have traditionally been considered tumor suppressors based on the frequent loss of GJIC and lower connexin expression in tumor cells compared to non-neoplastic tissue. For this reason, connexin 43 (Cx43) has been proposed to suppress the growth of glioblastoma (GBM), the most common primary malignant brain tumor. However, recent data suggest that this tumor-suppressive effect is context dependent and that connexins can also function in a tumor-promoting role. Using next-generation sequencing techniques, we found Cx43 expressed at high levels in a panel of GBM patient-derived xenograft (PDX) CSCs and that these models rely on Cx43 for their survival and self-renewal. Mechanistically, depletion of Cx43 led to a dramatic loss of c-MYC expression through reduced phosphorylation of its upstream mediator WNK lysine-deficient protein kinase 1 (WNK1). Depletion of WNK1 phenocopied knockdown of Cx43 and reduced MYC protein and mRNA as well as tumor growth in vivo. Together, this work defines a novel signaling axis downstream of gap junction protein expression that promotes tumor growth and cancer stem cell phenotypes in GBM. Due to the difficulty in targeting both Cx43 and MYC, the identification of intermediate targetable signaling nodes may lead to improved therapies for patients with GBM.

EXTH-37. ALLOSTERIC MITOCHONDRIAL CLPP AGONIST ONC206 ALTERS STRESS RESPONSE, METABOLIC AND EPIGENETIC PROFILES TO ELICIT ANTI-CANCER EFFICACY IN HIGH-GRADE GLIOMAS

Abstract Dordaviprone (ONC201) is a dopamine receptor D2/3 (DRD2/3) antagonist and allosteric ClpP agonist that has demonstrated durable responses in recurrent H3 K27M-mutant glioma patients. ONC206, a derivative of ONC201 with nanomolar potency that also targets DRD2/3 and ClpP, is in Phase I trials for central nervous system tumors. We characterized target relevance, human ClpP binding, in vitro efficacy, downstream signaling and response determinants associated with ONC206 in high-grade gliomas (HGG). Co-crystallization with ClpP that assembles as a tetradecameric complex revealed an allosteric ligand interaction with distinctions in the ONC206-ClpP overall conformation relative to the ONC201-bound or apo complex, including an increase in the resolved pore size and decreased complex height. ONC206 was more potent than ONC201 in cell-free human ClpP casein/peptide degradation assays). Accordingly, HGG cell lines and patient-derived cells exhibited increased sensitivity to ONC206 relative to ONC201 and generally required a shorter duration of exposure. Unlike DRD2, CRISPR-mediated ClpP knockout in HGG cell lines blunted ONC206 sensitivity. Metabolomics revealed elevated α-ketoglutaric acid, α-hydroxyglutaric acid, and glutaric acid. Proteomics indicated inhibition of multiple mitochondrial pathways including OXPHOS and TCA cycle. TCA cycle enzyme α-ketoglutarate dehydrogenase (KGDH) was inhibited in a ClpP-dependent manner. Western blotting showed downregulated ClpX, and upregulated ATF4/CHOP, H3 K27me3 and H3 K36me3. Next-generation sequencing of acquired resistance cell lines, which exhibited cross-resistance to ONC201 and ONC206, revealed diverse ClpP missense mutations distributed across various monomer interfaces. Both intrinsic and well as ONC206-stimulated ClpP proteolysis were mitigated by these mutations, which was congruent with an absence of the tetradecameric assembly. These ClpP mutations recapitulated resistance and, conversely, wildtype ClpP overexpression restored sensitivity. Thus, allosteric ClpP agonism is a key determinant of ONC206 activity in HGG by altering stress response, metabolic, and epigenetic profiles.