bluuebunny/crossref_metadata_2025 huggingface dataset:
BIOM-28. PROGNOSTIC BIOMARKERS IN GLIOBLASTOMA PATIENTS TREATED WITH TEMOZOLOMIDE
Authors: Zhang Shuodan , Mcmillan Nadia , McGuinness Matthew , Trudeau Stephen , Uhlmann Erik
Published: November 2023

OBJECTIVE

Identifying blood biomarkers that predict favorable outcomes for patients with glioblastoma treated with temozolomide (TMZ).

BACKGROUND

Glioblastoma is the most aggressive primary brain tumor. The standard of care is maximum safe resection, then radiation plus oral TMZ, followed by six months of cyclic oral TMZ. Previous studies have revealed that TMZ-induced myelosuppression is associated with improved overall survival.

METHODS

Adults with glioblastoma who completed radiation therapy and received temozolomide for at least 6-months at Beth Israel Deaconess Medical Center between 2018-2021 were identified. Baseline and nadir white blood cell count, absolute neutrophil count, absolute lymphocyte count, red blood cells, and platelets were collected retrospectively. Additionally, IDH1 R132H, MGMT promoter methylation, progression free survival (PFS), and overall survival (OS) were also collected retrospectively. Data was combined with that of Vaios et al. and Ho et al. Kaplan-Meier analysis and multivariate testing were performed.

RESULTS

Leukopenia is associated with increased overall survival (p = 0.008) and progression free status (p = 0.02). Grade 3 thrombocytopenia is associated with decreased PFS (p = 0.0005) and OS (p = 0.0006). Grade 1 & 2 lymphopenia is associated with increased progression free survival (p = 0.01 and p = 0.03). Lower baseline absolute lymphocyte count is associated with improved OS (p = 0.007). Higher baseline absolute neutrophil count is associated with worse OS (p = 0.002).

CONCLUSION

Cytopenia may serve as a prognostic biomarker for OS and PFS. Leukopenia may reflect adequate temozolomide dosing, anti-tumor activity, or changes in the tumor micro-environment. Peripheral biomarker monitoring may improve prognostic stratification and lead to personalized treatment with improved outcomes.
BIOM-30. CELL LINE STUDY OF NUCLEOSOME-BASED BIOMARKERS IN THE DIAGNOSIS AND DETECTION OF RELAPSES IN GLIOBLASTOMA
Authors: Decarpentrie Jonathan , Van den Ackerveken Priscilla , Govaerts Adrien , Lobbens Alison , Beine Edeline , Donis Nathalie , Herzog Marielle , Douxfils Jonathan
Published: November 2023

Currently, glioblastoma (GBM) diagnosis and monitoring rely on neuroimaging and histopathological confirmation. However, overall survival has not improved in last decades due to therapeutic failure and to a lack of biomarkers for relapses’ detection. Liquid biopsies (i.e. blood or CSF) using nucleosomes-containing-histone post-translational modifications (PTMs) have the potential to become valuable biomarkers for diagnosis and monitoring GBM. Four glioblastoma cell lines (SF-126, U-87MG, U-118MG, and U-138MG) compared to a healthy microglia cell line (HMC3) and other solid cancer cell lines including pancreas (MIA-PaCa-2), liver (HepG2), and cervix/uterus (HeLa) have been analyzed to identify their epigenetic profile. Nucleosomes were extracted and analyzed with the Nu.Q® Discover immunoassays platform addressing 13 histone-PTMs. Quantitative results of PTMs expression were normalized on quantification of total H3.1-nucleosomes. The immunoassay identified three candidate biomarkers compared to control cell lines (n = 4): citrullinated-histone-H3 (H3R8Cit) (control = 4.68%; GBM = 13.88%; p< 0.05), phosphorylation of H3S10 (H3S10Ph) (control = 2.31%; GBM = 5.57%; p< 0.05), and a trend in H3K4Me2 elevation (control = 0.43%; GBM = 1.07%; p = 0.066) in GBM cell lines (n = 4). These results were confirmed by western blot. Moreover, principal component analysis revealed a segregation between GBM and control cells, mainly driven by these three PTMs. Moreover, to demonstrate the capacity of the Nu.Q® assays to quantitatively monitor PTMs, GBM cell line SF-126 (n = 3) was treated with an EZH2 inhibitor (responsible of H3K27 methylation) (iEZH2). After 24 hours and 48 hours of exposure, the expression level of H3K27Me3 –expressed as ratio of total nucleosomes– was decreased by 32% (control = 14.67%±0.58%; iEZH2= 10.00%±1.73%; p< 0.05) and 41% (control = 15.33%±1.53%; iEZH2= 9.00%±0.00%; p< 0.05), respectively. Those results were also confirmed by western blot analysis. In conclusion, we identified three specific in vitro epigenetic-based marks of GBM and demonstrated that the Nu.Q® technology is a valuable tool to monitor the degree of PTMs expression.
BIOM-32. EVS CARRYING TENASCIN-C ENABLE TUMOR-SPECIFIC MUTATION ANALYSIS IN GLIOBLASTOMA PATIENTS
Authors: Salviano-Silva Amanda , Maire Cecile , Wollmann Kathrin , Reimer Rudolph , Neumann Julia , Dottermusch Matthias , Schüller Ulrich , Gempt Jens , Heiland Dieter Henrik , Westphal Manfred , Lamszus Katrin , Ricklefs Franz L
Published: November 2023

Extracellular vesicles (EVs) transport biological and specific information from tumors into the bloodstream, enabling non-invasive detection of tumor material and disease monitoring. Based on a proteomics screen, we performed immunophenotyping of eight glioma-related antigens (tenascin-C (TNC), integrin-beta 1 (ITGB1), profilin-1 (PFN1), CD44, CD133, GPNMB, HLA-II, SPARC) and tetraspanins (CD9, CD63 and CD81) in plasma EVs from GBM patients (before and after surgery (n = 38)), from matched GBM relapse patients (n = 11), and from healthy donors (HD, n = 12) using imaging flow cytometry. Double-positive TNC+/CD9+ EVs showed the strongest differences per mL of plasma in primary (FC = 7.6, p< .0001, ROC analysis AUC = 81%) and relapsed GBM (FC = 16.5, p< .0001; AUC = 90%) compared to HD subjects. High TNC signals were also observed in GBM-EVs by immunogold electron microscopy compared to HD-EVs. In paired analysis, TNC+/CD9+ EVs showed a 3.9-fold decrease after tumor removal (p< .001) and re-increased at GBM recurrence in these patients (FC = 8.4, p< .05; AUC = 84%). In tissue samples, TNC levels were 5.4-fold higher in GBM patients than in non-neoplastic cortex controls (p< .01) measured by immunohistochemistry and correlated positively with plasma TNC+/CD9+ EV levels in RTK-I/II GBM patients (r = 0.42, p< .05). Accordingly, spatial transcriptomics of GBM tissue sections revealed that TNC is specifically overexpressed in GBM cells. Furthermore, magnetic sorting of TNC in plasma EVs allowed detection of GBM-specific TERT*C228T mutations by digital droplet PCR. Mutated TERT DNA was enriched in TNC+ EVs (n = 13) compared to TNC- EVs (FC = 110, p< 0.0001), total EV DNA (FC = 36.7, p< 0.01), and cfDNA (FC = 7.2, p < 0.05). In conclusion, we identified TNC as a biomarker in circulating EVs from GBM patients that can be isolated and used for tumor-specific mutation analysis.
BIOM-34. NON-NEOPLASTIC PLASMA EV SUBPOPULATIONS CORRELATE WITH TUMOR SIZE AND OVERALL SURVIVAL IN GLIOBLASTOMA INDEPENDENT OF STEROID USAGE
Authors: Aibaidula Abudumijiti (Zack) , Bauman Megan , Ghaith Abdul Karim , Bouchal Samantha , Parney Ian
Published: November 2023

INTRODUCTION

Extracellular Vesicles (EVs) are lipid bilayer particles present in almost all biofluids and carry tumor-specific molecular cargo. Plasma EVs are promising sources of biomarker discovery for glioblastoma (GBM). The clinical relevance of non-neoplastic plasma EVs in GBM remains unclear. In this study, we determine if associations exist between plasma EV subpopulation frequencies in GBM and clinical factors.

METHODS

Platelet-free plasma samples of 63 GBM, 43 Grade 2 glioma, and 45 healthy donors were included. All glioma plasma samples were collected before surgery. Plasma EV subpopulation frequency based on surface expression of EV-associated tetraspanins (CD9/CD81/CD63) and markers of cell origin (CD45/CD41a/CD11b/CD31) was determined by spectral flow cytometry. Clinical information including age, surgery, MGMT methylation status, corticosteroid usage, tumor volume, and overall survival data was recorded.

RESULTS

Compared to normal donors, GBM patients had increased CD9+, CD63+, CD81+, and CD11b+ EVs but decreased CD41a+ EVs. GBM patients also had the highest frequency of myeloid-derived CD9+CD11b+ EVs (20.17%) followed by grade 2 gliomas (13.78%) and normal donors (2.76%, p< 0.001). More GBM patients (29/63, 46.0%) received preoperative corticosteroids than grade 2 glioma patients (9/43, 20.9%; p = 0.008). Corticosteroid use was not associated with differences in EV subpopulation frequencies in GBM or grade 2 glioma patients. Pearson correlation analysis showed weak but statistically significant correlations between GBM total tumor volume and CD9+ EVs (p = 0.014), CD41a+ EVs (p = 0.037), and CD9+/CD63-/CD81- EVs (p = 0.007). In GBM patients, younger age (< 60 yrs, p = 0.01), gross total resection (p = 0.02), MGMT methylation (p = 0.002), high CD9+ EV expression (p = 0.011), low CD9+CD63-CD81- EV expression (p = 0.009) and low CD9+CD11b+ EV expression (p = 0.006) correlated with longer overall survival.

CONCLUSION

Differentially expressed non-neoplastic plasma EV subpopulations have significant clinical relevance in GBM including association with tumor volume and predicting survival. Validation of those markers in larger cohorts could reveal new prognostic biomarkers for GBM.
BIOM-37. EXTRACELLULAR VESICLES AS A PHARMACODYNAMIC REPORTER IN GLIOBLASTOMA
Authors: DeLuca Valerie , Tang Nanyun , Hao Yue , Raju Anjali , Shaffer Charles , Riviere-Cazaux Cecile , Burns Terry , Sarkaria Jann , Parney Ian , Pirotte Patrick , Van Keuren-Jensen Kendall , Berens Michael
Published: November 2023

Development of a non-invasive test that can measure drug delivery and target engagement in GBM represents an opportunity to enhance knowledge during early phase clinical trials of a new compound’s pharmacological suitability and potential efficacy. We present here data supporting that the cargo within extracellular vesicles (EV) can potentially serve as a liquid biopsy that reports on the pharmacodynamics of GBM therapeutics. We treated short-term PDX cultures with vehicle, temozolomide, MLN4924, or arsenic trioxide. EVs were isolated from culture supernatants using size exclusion chromatography and then analyzed by mass spectrometry (proteomics) and RNA sequencing to characterize and compare cargo profiles for each condition. We found that at the proteome level, FDFT1 and FARSB were detected only in temozolomide EVs across all cell lines. In addition, there were drug-agnostic indications of exposure, including decreased abundance in all treated EV conditions of SLC3A2, C3, PLG, and A1BG, and increased abundance of GSTP1. These cargo changes were further validated by western blotting and ExoView immunofluorescence on isolated EVs. At the EV-transcriptome level, depending on treatment, there were shared alterations in EV cargo between models in the expression of long non-coding RNAs, genes that are overrepresented in biological processes such as cytoplasmic translation and cellular macromolecule biosynthesis, and circular RNAs. Thus, these results indicate that EV cargo post-treatment may be a novel pharmacodynamic reporter in GBM. To allow immunoprecipitation of patient GBM-derived EVs with the selective interrogation, the potential biomarkers, including PTPRZ1, B7H3, IL13RA2, and EGFR were screened across isolated EVs from both pre-clinical and clinical patient samples. Future efforts are focused on evaluating immunoprecipitated GBM-EVs from longitudinally collected patient biofluids. Overall, we anticipate that the results of this study will lead to development of a clinical test that reflects BBB penetration and tumor response, which will likely aid in novel drug development efforts.
BIOM-38. MET FUSIONS AND SPLICING VARIANTS IS A STRONG ADVERSE PROGNOSTIC FACTOR IN ASTROCYTOMA, IDH-MUTANT
Authors: Zhang Kenan , Liu Lingyu , Zhao Zheng , Chen Jing , Liu Xing , Jiang Tao
Published: November 2023

BACKGROUND

Astrocytoma, IDH-mutant is currently characterized by both histological and molecular features according to the WHO CNS5 classification. However, there were still patients with IDH-mutant lower-grade astrocytomas (LGAs, CNS WHO grade 2 and 3) who suffered poor survival. Our previous research demonstrated the enrichment and predictive role of MET fusions and/or splicing variations (MET F/SVs) in previously classified secondary IDH-mutant glioblastoma, which could be a potential complement to the existing grading system.

MATERIALS

We herein presented descriptive, Kaplan-Meier, multivariable Cox regression outcome analyses, and DNA methylation profiling cluster analysis in a large cohort of astrocytomas, IDH-mutant.

RESULTS

A total of 427 astrocytomas, IDH-mutant were included. 28/427 (6.56%) patients were defined as carrying MET F/SVs, including 9/261 (3.45%) LGAs and 19/166 (11.45%) IDH-mutant high-grade astrocytomas (HGAs, CNS WHO grade 4). The distribution of MET F/SVs was significantly different between LGAs and HGAs (p = 0.0011). Kaplan-Meier analysis of PFS and OS confirmed MET F/SVs was significantly associated with inferior survival irrespective of CNS WHO grade. Multivariable Cox regression analysis with PFS and OS as the endpoint showed MET F/SVs were independently associated with worse survival (p = 0.006 for PFS and p < 0.0001 for OS). Moreover, we collected 159 astrocytomas, IDH-mutant with DNA methylation profiles for further analysis. The included patients were classified into the methylation class MC astrocytoma, IDH-mutant in version 12.5 of the Heidelberg brain tumor classifier. On the t-distributed stochastic neighbor embedding (t-SNE) projection, the IDH-mutant astrocytoma with MET F/SVs was more clustered in high-grade astrocytoma (p = 0.017).

CONCLUSION

Collectively, we demonstrated the utmost relevance of MET F/SVs as an adverse prognostic factor in IDH-mutant astrocytoma independent of the current WHO CNS grading system. We proposed that the LGAs with MET F/SVs should be designated into CNS WHO grade 4.
BIOM-40. THERABIOME-GBM: THERAPEUTIC OUTCOMES RELATED TO GUT MICROBIOME IN GLIOBLASTOMA (GBM) PATIENTS RECEIVING CHEMO-RADIATION: A PROSPECTIVE OBSERVATIONAL STUDY
Authors: Ng Terry , Alshamsan Bader , Nguyen Thanh , Butcher James , Stober Carol , Vandermeer Lisa , Nicholas Garth , Stintzi Alain , Nair Vimoj
Published: November 2023

PURPOSE

A major barrier to improving outcomes in glioblastoma multiforme (GBM) is overcoming a heavily immunosuppressed tumor and systemic environment, as demonstrated by several negative phase III randomized studies of nivolumab in both newly diagnosed and recurrent GBM. In several mouse avatar and human studies across several tumor cohorts including in a humanized microbiome GBM model, re-establishing a new gut microbiome (GM) via fecal transplantation led to tumor responses to PD-1/PD-L1 inhibition. A recent scoping review confirmed a paucity of GM clinical data of a uniform GBM cohort with adequate longitudinal GM sample collection. We are conducting a single-center prospective observational study to establish the feasibility of collecting stool samples throughout the time course of GBM treatment and upon recurrence, and to understand the gut microbiome dynamics of newly diagnosed IDH-wild type (WT) GBM patients.

METHODS

Patients with newly diagnosed unifocal IDH-1 R132H WT WHO grade 4 GBM, ECOG 0-2, at least 70% tumor resected with plan to receive temozolomide-radiation (TMZ-RT) and adjuvant TMZ +/- Tumor Treating Fields are eligible. Stool samples will be collected prior to TMZ-RT, 4 weeks after TMZ-RT, after adjuvant TMZ, and upon recurrence. The primary endpoint is feasibility, defined as stool sample obtained pre-RT, post-RT (pre-adjuvant TMZ), and at time of recurrence in ≥ 70% of enrolled participants, 75% of target sample size enrolled within 2 years, and stool sample volume and quality sufficient for analysis in ≥ 75% of collected samples. Secondary endpoints include progression-free survival (PFS) in pre-defined GM subgroups, GM taxonomy and diversity in late vs. early progressors, and GM differences in patients with and without post-RT necrosis. Age, sex, race, concurrent medication, corticosteroid use, and diet will be carefully accounted for. Patient reported outcomes using EORTC-QLQ-BN20 and QLQ-C30 will be collected at baseline, 3-, 6-, 9-, and 12-months.
BIOM-45. GLIOMA CSF PROTEOMICS FOR PHARMACODYNAMIC BIOMARKER AND THERAPEUTIC TARGET DISCOVERY
Authors: Riviere-Cazaux Cecile , Carlstrom Lucas , Munoz-Casabella Amanda , Andersen Kari , Ikram Samar , Warrington Arthur , Burns Terry
Published: November 2023

Cerebrospinal fluid (CSF) is emerging as an abundant source of candidate biomarkers, including proteins, for gliomas. Longitudinal CSF access may enable insights into treatment response and resistance. To that end, we have established a growing biobank of CSF samples, many of which have been obtained longitudinally via CSF access devices, such as Ommaya reservoirs, through our ongoing biomarker discovery clinical trials (NCT04692337, 04692324). We have previously presented our initial data demonstrating a conserved proteomic signature of HGG that decreased with resection and increased with recurrence. Since then, samples have been acquired throughout individual patients’ treatment course toward our goal of identifying CSF-derived biomarkers of treatment response. CSF samples obtained prior to, during, and throughout treatment underwent global proteomics analysis via the SomaScan® assay by Somalogic. Fold-changes for each protein were calculated between different timepoints and ranked for each comparison of interest. Treatment with pembrolizumab alone results in increased IL-23 (5.01x), IL-12 (4.25x), lymphocyte activation gene 3 protein (LAG3) (3.26x), and granzyme A (2.93x), suggesting potential for combination with multiple agents. Chemoradiation, with or without pembrolizumab, consistently downregulated NAD kinase, acetyl CoA transferase, and Bcl-2 and upregulated proteins related to neuronal signaling, such as neurexins and cerebellins, in addition to LAG-3, suggesting potential for synergy between chemoradiation and anti-LAG-3 antibodies currently in clinical trials. Interestingly, pembrolizumab and chemoradiation decreased MMP-12 and MMP-9 abundance as compared to chemoradiation alone (0.25x vs. 18.5x; 0.54x vs. 4.99x). Finally, bevacizumab treatment consistently increased VEGF-A (7.32x), VEGF-B (2.21x), and MMP-3 (2.99x) abundance, while decreasing the presence of our previously reported plasma-associated protein signature in CSF. In conclusion, the global CSF proteome can be longitudinally accessed to evaluate response to standard-of-care and experimental therapies. Further studies are ongoing to identify signatures of therapeutic resistance and to assess the impact of novel immunotherapeutic combinations on the CSF proteome.
BIOM-47. CLINICAL AND MOLECULAR FEATURES OF PATIENTS WITH LI-FRAUMENI SYNDROME AND GLIOMA: AN MD ANDERSON CANCER CENTER EXPERIENCE
Authors: Lam Keng , Kamiya-Matsuoka Carlos
Published: November 2023

BACKGROUND

Li-Fraumeni Syndrome (LFS) predisposes patients for cancer, including gliomas, due to pathogenic TP53 germline variants. Little is known about their treatment course, molecular markers, and prognosis.

METHODS

This is a retrospective study of patients with diagnosis of LFS and glioma at MD Anderson Cancer Center from 2013 to 2023. Data collected includes next generation sequencing and treatment history. We analyzed descriptive statistics and Kaplan-Meier method to report their clinical characteristics and median progression-free survival (mPFS), defined as the time from initial diagnosis to first recurrence.

RESULTS

14 patients with LFS had diagnosis of glioma, with half of them as the first manifestation of cancer. There were 8 IDH-mutant gliomas (all WHO grade 2 astrocytoma), 4 IDH-wildtype WHO grade 4 glioblastoma, and 2 gliomas with no molecular markers (WHO grade 3 astroblastoma and astrocytoma). Only IDH1 mutations were identified: R132H (n = 4), R132S (n = 2), and R132L (n = 2). ATRX status was tested in 12 gliomas, 8 with retained protein expression. ATRX co-mutation in IDH-mutant astrocytoma occurred in 38% of cases. TP53 status was tested in 12 gliomas, with 6 having positive expression on immunostaining. MGMT methylation status (n = 8) was detected in 2 IDH-mutant gliomas whereas all glioblastomas were unmethylated. For treatment, 7 patients with low-grade glioma opted for observation after initial diagnosis despite 5 being considered high-risk for recurrence (subtotal resection/ > 40 years of age). The mPFS for low-risk and high-risk low-grade astrocytoma on observation was 29.1 months and 28.5 months respectively. Based on IDH status, the mPFS for glioblastoma and IDH-mutant grade 2 astrocytoma was 6.4 months and 25.5 months respectively.

CONCLUSIONS

LFS patients may have glioma with higher incidence of non-canonical IDH1 mutations, lower likelihood of ATRX and IDH1 co-mutations, and less likely to be treated upfront after initial diagnosis despite having high risk profile for low grade glioma.
BIOM-49. FGFR ALTERATIONS IN GLIOMAS; CLINICAL, RADIOGRAPHIC AND MOLECULAR CHARACTERIZATION
Authors: Hill Colin , Iavarone Antonio , Tajera Dalissa , de la Fuente Macarena
Published: November 2023

BACKGROUND

FGFR-TACC is the most prevalent type of gene fusion in adult gliomas. It is present in 3-6% of IDH WT (wild-type) gliomas. Due to their histopathological and molecular features, it has been suggested that FGFR3-TACC3 fusions (F3T3) gliomas represent a unique entity among IDH WT gliomas. F3T3 are potent oncogenes that confer sensitivity to FGFR inhibitors. Preliminary data showed promising results of these alterations as actionable targets by FGFR inhibitors.

METHODS

We retrospectively analyzed molecular data on 912 glioma patients treated at our institution from 2014 to 2023.

RESULTS

We identified 22 gliomas (2.5%) with FGFR alterations. Median age was 60 years, 14 were female. 14 patients (64%) self-identified as Hispanic. 18 had a diagnosis of glioblastoma, 3 had other gliomas, and one had a low-grade neuroepithelial neoplasm. Ten patients had tumor location in the temporal lobe, 6 frontal, 5 parietal, 3 occipital, 1 multifocal, and 1 basal ganglia. 19 patients had enhancing tumors, while three were non-enhancing. The most common FGFR alteration was FGFR3-TACC3 fusion in 14/22 patients, 6 had FGFR mutations (either single base or amino acid substitutions), one had FGFR-LMNB2 fusion, and one had FGFR3 amplification. 86.4% of our patient cohort (19/22) had a confirmed TERT promoter mutation, while 100% of GBM patients with known TERT promoter status had an associated TERT promoter mutation. 10/22 had CDKN2A/B alterations. One patient had pathology-proven systemic metastases.

CONCLUSIONS

FGFR3-TACC3 fusions (F3T3) gliomas represent a unique entity among IDH WT gliomas. In our cohort, 100% of the patients with F3T3-positive glioblastomas also had an associated TERT promoter mutation. To our knowledge, this is one of the larger cohorts showing the association between FGFR3 and TERT promoter alterations in glioblastomas.

RESULTS

will be validated through the upcoming clinical trial ETCTN 10559, including tumor evolution and mechanisms of resistance.
BIOM-58. ZERO-WASTE MOLECULAR DIAGNOSTICS FROM BIOPSY NEEDLE WASHES
Authors: Wadden Jack , John Vishal , Niculcea Michael , Kong Seongbae , Aittaleb Rayan , Cartaxo Rodrigo , Verbal Kait , Bruzek Amy , Magge Suresh , Yadavilli Sridevi , Erez Nir , Furth Noa , Shema Efrat , Marapudi Neena , Heth Jason , Al-holou Wajd , Garton Hugh , Nazarian Javad , Koschmann Carl
Published: November 2023

BACKGROUND

Brain tumor classification has increasingly integrated molecular diagnostics into official criteria. However, biopsy tissue is limited due to the small quantity acquired in eloquent areas of the brain and is, therefore, usually earmarked for histology. We have identified that the fluid used to wash biopsy needles contains excess tumor material that can be diagnostically useful. Characterization of this non-zero-sum waste material provides an opportunity for improvement in the diagnosis and treatment for brain tumor patients.

METHODS

For suspected or established high grade brain tumor biopsies (n = 37 across a multi-institutional cohort at submission), neurosurgeons were instructed to save fluid used to wash biopsy needles. Samples were processed and characterized in a variety of methods to establish diagnostic utility including (1) cell-free DNA isolation and targeted panel sequencing (n = 13), (2) isothermal amplification of cell-free DNA from raw wash water for rapid diagnostics (n = 2), (3) cell-line generation (n = 24) and, (4) cell-free mutant protein capture and quantification (n = 9).

RESULTS

(1) 13 wash samples were considered for cf-tDNA extraction and Illumina TruSight Oncology 500 panel sequencing (with extracted DNA ranging from 20ng-40ug). In 2 returned cases (11 pending), concordance was 100% with clinical targeted panel sequencing where panels overlapped. (2) One case suspected of harboring H3K27M was subjected to targeted, loop-mediated isothermal amplification and Oxford Nanopore real-time sequencing, confirming an H3.3K27M diagnosis (11.8% allele fraction) within 1hr35min of receipt of sample from the operating room. (3) 5 cases resulted in primary cell culture generation (4) A novel single-molecule mutant protein quantification technique was performed on 9 cases.

RESULTS

from all analyses and concordance with outstanding sequencing will be presented.

CONCLUSION

We demonstrate that biopsy byproducts contain enough high-quality tumor-derived material to perform a variety of useful diagnostics with the potential to have immediate translational impact on standard of care.
BIOM-60. CONCATEMERIC CONSENSUS ERROR CORRECTED SEQUENCING (CCEC-SEQ) APPROACH FOR PLASMA CELL-FREE TUMOR DNA-BASED TREATMENT RESPONSE MONITORING IN PATIENTS WITH BRAIN TUMORS
Authors: Wadden Jack , John Vishal , Aittaleb Rayan , Koschmann Carl
Published: November 2023

BACKGROUND

Diffuse midline gliomas (DMG) are aggressive and universally fatal with a median survival rate of only 9-12 months. MRIs can be difficult to interpret due to diffuse disease and radiation-induced swelling. Recent work has shown that liquid biopsy of cell-free tumor DNA (cf-tDNA) in patient plasma using digital droplet PCR (ddPCR) might help supplement radiographic monitoring, and enable more quantitative, low-cost, rapid tests. Nanopore sequencing has been employed to quickly and affordably measure and track treatment response in DMG patient cerebrospinal fluid (CSF) where cf-tDNA variant allele fractions are high ( >1%). However, plasma cf-tDNA signal is much lower than CSF (typically < 0.5%), and no platforms have demonstrated the feasibility of quickly (< 1day) sequencing brain tumor cf-tDNA in plasma samples to our knowledge.

METHODS

To solve these problems, we have developed a same-day, multiplexed, concatemeric consensus error corrected (CCEC-Seq) assay approach for Nanopore sequencing. We combine multiplex PCR and loop-mediated isothermal amplification (LAMP) to capture short cf-tDNA fragments and concatemerize them. Sequenced concatemer segments should agree, and allow informatic consensus error correction, reducing Nanopore error rates.

RESULTS

We generated a multiplex (n = 8) DMG hotspot loci panel, which was able to reduce sequencing false positive call rates by 3.1x-10.8x. We were able to employ the CCEC-Seq technique on serial plasma samples from a patient with DMG with H3.3K27M mutation undergoing treatment with ONC201 with results comparable to a previously validated ddPCR assay. Ongoing work will apply this multiplex CCEC-Seq panel to a larger cohort of serial DMG plasma samples.

CONCLUSION

To our knowledge, this is the first demonstration of same-day, sequencing-based, liquid biopsy protocol for a plasma sample from a brain tumor patient. This approach has the potential to greatly reduce resource requirements, and ease of operation for liquid biopsy for DMG and other brain tumor disease monitoring.
BIOM-61. EXPLORING HOMOLOGOUS RECOMBINATION DEFICIENCY IN PEDIATRIC HIGH-GRADE GLIOMAS
Authors: Xu Ke , Savage Evan , Myers Jason , Rampersaud Evadnie , Hui Yawei , Chang Ti-Cheng , Wu Gang , Tinkle Christopher , Shelat Anang
Published: November 2023

Homologous recombination deficiency (HRD) has been extensively studied in breast cancer and ovarian cancer given its correlation with sensitivity to Poly (ADP-ribose) polymerases (PARP) inhibitors. In these cancers, HRD is most frequently caused by biallelic inactivation of BRCA1/2 and tumors often exhibit ‘genomic scars’ such as elevated HRD.sum score and specific mutational signatures, including specific single nucleotide variant (SNV), small insertion/deletion (Indel), and structural variant (SV) signatures. Despite its clinical relevance, HRD has not been systematically studied in pediatric cancers and its therapeutic predictive value is unknown. Here, we evaluated HRD in 255 tumor-normal paired whole-genome-sequenced (WGS) samples of pediatric high-grade glioma (pHGG) and 23 tumor-normal paired WGS samples of breast cancer (BC) as comparison. We found that 9% of the pHGG samples have HRD.sum score greater than 40, an indicator of HRD in several adult cancers. However, while we identified HRD-related SNV signature SBS3 and SV signature RS5 in some of the pHGG samples, HRD-related Indel signature ID6 was not identified. We also evaluated two composite scores, namely, HRDetect and CHORD, on our samples. While virtually all BC samples with BRCA1/2 loss were predicted to be HRD by both scores, none of the pHGG samples was predicted to be HRD by either score. We profiled somatic and germline variants from over 500 DNA damage response genes in the pHGG samples and identified TP53 as the most frequently mutated genes with biallelic loss, whereas biallelic loss of BRCA1/2 was not found. Overall, our study suggests that HRD exists in a fraction of pHGG samples, but is driven by inactivation of genes other than BRCA1/2, resulting in a different set of genomic scars compared to those in BCs. Functional analysis of sensitivity to PARP inhibitor and related DNA damage inhibitors is ongoing to evaluate potential therapeutic relevance of these genomic scars.
BIOM-65. NEW BIOMARKERS FOR THE DIAGNOSIS OF PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA IN CEREBROSPINAL FLUID: AN EXPLORATORY MULTICENTER RETROSPECTIVE COHORT STUDY
Authors: van Rooij Josephus , Wessels Peter , Delemarre Eveline , Meek Bob , Ruven Henk , Seute Tatjana , Minnema Monique , Nierkens Stefan , Snijders Tom
Published: November 2023

BACKGROUND

Primary central nervous system lymphoma (PCNSL) is a rare form of non-Hodgkin lymphoma which may be treated with curative intent. Diagnosis often requires a brain biopsy, which is associated with complications and delay of diagnosis. Cerebrospinal fluid (CSF) analysis can currently provide a diagnosis (without the need for a biopsy) in only a minority of PCNSL patients. The aim of this study is to identify new potential biomarkers in CSF for patients with clinical suspicion of PCNSL, to improve the diagnostic yield of CSF analysis.

METHODS

CSF samples were analyzed from a retrospective cohort that included patients with clinical suspicion of PCNSL at two Dutch hospitals between August 2020 and October 2021. First-step analysis consisted of targeted multiplex biomarker analysis using a proximity extension assay (Olink), allowing the simultaneous analysis (relative expression levels) of 184 protein markers in a small CSF volume. To validate the discovered biomarkers in a quantitative manner, a second analysis was performed in a subgroup of patients with a bead-based multiplex immune-assay (Luminex).

RESULTS

A total of 76 patients were included, of whom 11 patients (14%) were diagnosed with PCNSL. The mean age was 62 years (range 22-83 years), and 62% of the patients were male. In the primary analysis, 31 markers showed significant differences (p< 0.05) between PCNSL patients and controls. After correction for multiple testing, 6 markers remained significant, including the known chemokine marker CXCL13, as well as new markers. The quantitative analysis confirmed significant differences (p< 0.05) for 3 markers: CXCL10, programmed cell death protein 1 (PDCD1) and FAS natural ligand (FASLG).

CONCLUSION

We identified a novel panel of potential diagnostic markers in CSF for the diagnosis of PCNSL. The diagnostic value of these markers will be validated in an ongoing multicenter prospective study.
BIOS-02. RECAPITULATE TUMOR DIVERSION AND SPATIAL PATTERNING IN GLIOBLASTOMA BACKGROUND
Authors: Lo Wei-Lun , Khoa Hoang Dang Ta , Chuang Jian-Ying
Published: November 2023

BACKGROUND

Intra-tumoral heterogeneity of glioblastoma multiforme (GBM) drives aggressive behavior and intrinsic therapeutic resistance. To investigate heterogeneity, high-resolution sequencing techniques provided us to feature tumor molecular subtyping and simulate differentiation. In our study, we proposed to dissect tumor heterogeneity in different anatomical regions by single cell transcriptome analysis. Material and Method We performed single-cell RNA sequencing on 10675 adult IDH-wild type GBM cells from six pairs of tumor core (central necrotic part of tumor) and tumor margin (junction between tumor and brain). Subtyping following RNA velocity was performed to investigate tumor diversity. Result Four subgroups of tumor cells (OPC-like (oligoprogenitor cell), NPC-like (neuroprogenitor cell), AC-like (astrocyte), and MES-like (mesenchymal) )were identified. The higher cell population of OPC/NPC at tumor margin and AC/MES at tumor core was found. The RNA velocity analysis showed the major diversity order was OPC at margin, OPC at core, NPC at margin, and AC with MES at core.

CONCLUSION

We explore the spatial pattern of tumorigenesis of GBM that OPC at margin part could be the tumor initiating cell. Further investigation of biological characteristics of OPC could be the strategies to block tumor differentiation and progression.
CNSC-01. THE ROLE OF SEROTONERGIC RECEPTOR, HTR2B, ON MYELOID DERIVED SUPPRESSOR CELLS IN THE BRAIN METASTATIC ENVIRONMENT
Authors: Iyer Mukund , Das Diganta , Nakamura Brooke , Sedighi Saman , Reed Max , Pakravan Alexa , Shah Priya , Jang Julie , Baugh Aaron , Gonzalez Edgar , Torres Evanthia Roussos , Neman Josh
Published: November 2023

In the United States, greater than 12% of cancer patients develop brain metastases (br-mets) and median survival remains an abysmal 4-6 months. Throughout the metastatic cascade, tumor cells attract and reprogram stromal cells, such as myeloid derived suppressor cells (MDSCs), which support cancer growth by promoting tissue remodeling, EMT, invasion, immunosuppression, angiogenesis. Once tumor cells have breached the brain’s CNS defenses, infiltrating immune cells can penetrate the glia limitans and enter the CNS parenchyma to help establish the brain metastatic niche. However, there is scant understanding of MDSC’s contribution to the brain metastatic microenvironment. The brain metastatic environment is particularly unique because CNS-resident cells such as microglia, astrocytes, choroid plexus, and neurons function as local mediators to alter invading immune cell plasticity and differentiation. We have demonstrated that MDSCs in the brain microenvironment respond to the neurotransmitter serotonin, upregulate the serotonergic receptor, HTR2B, resulting in increase pNF-κB and its associated signaling genes (IL-6, IL-1B, COX2, CXCL2, NOS2). Furthermore, HTR2B knockdown in MDSCs alters downregulates expression of pNF-κB signaling genes. Additionally, MDSCs in this environment support tumor cell proliferation while hindering their acquisition of neuronal traits. A better understanding of the role of HTR2B in brain metastasis may allow us to develop new treatment strategies for patients of this devastating disease.
CNSC-06. REDIRECTING IMMUNOSUPPRESSIVE GLIOMA MICROENVIRONMENT THROUGH PHARMACOLOGICAL INTERVENTION TARGETING NEURONAL ACTIVITY
Authors: Nejo Takahide , Krishna Saritha , Yamamichi Akane , Jimenez Christian , Lakshmanachetty Senthilnath , Young Jacob , Phyu Su , Watchmaker Payal , Hervey-Jumper Shawn , Okada Hideho
Published: November 2023

Recent single-cell RNA-sequencing analyses have revealed a strong association between glioma-neuronal circuit remodeling and regional immunosuppression. Subsequent preclinical investigations using syngeneic tumor models demonstrated that genetic disruption of circuit assembly genes, including Thrombospondin-1 (TSP-1/Thbs1) in GBM cells suppressed synaptogenesis, excitatory neurotransmitter ligand and receptor density in addition to neuronal activities while simultaneously restoring inflammatory responses, indicating glioma-neuron-immune cross talk. However, the underlying mechanisms and directions of causality remain unclear. Here we test the hypothesis that tumor-derived synaptogenic factors directly reprogram the tumor immune microenvironment (“model 1”). Alternatively, tumor intrinsic neuronal activity may indirectly regulate adaptive immunity (“model 2”). In this study, we performed in-vitro bone-marrow-derived macrophage (BMDM) reprogramming assays evaluating various stimulation conditions and in-vivo experiments evaluating the effect of neuronal activity-oriented therapy on the tumor-associated macrophages (TAMs). Our in-vitro assays demonstrated that, secreted factors from mouse neonatal cortical neurons (not tumor-derived synaptogenic factors) induced an anti-inflammatory, M2-like phenotype of BMDMs, corroborating the proposed model 2. This M2-inducing effect was inhibited when the neuronal activities were suppressed pharmacologically, using perampanel (PER), a noncompetitive inhibitor of neuronal AMPA-type glutamate receptors. Direct treatment of BMDMs with PER did not affect the polarization patterns, further supporting the M2-inducing effect of glutamatergic excitatory neuronal signals. In in-vivo syngeneic orthotopic tumor models, treatment with PER significantly prolonged survival compared to the non-treated group (27.5 [PER] vs. 23 days [control], p = 0.01). Flow cytometric analysis of brain-infiltrating leukocytes showed that TAMs isolated from PER-treated mice exhibited a more proinflammatory M1-like polarization compared to the control group (median M1/M2 ratio, 1.2 [PER] vs. 0.7 [control], p = 0.004), which explained the improved prognosis. Collectively, these findings unveil novel immunosuppression mechanisms induced by glioma-neuronal circuit remodeling and the resulting excitatory neuronal signals, and potential therapeutic vulnerabilities that could be targeted in combination with cancer immunotherapy.
CNSC-08. PROTEOGENOMIC ANALYSIS OF LONGITUDINAL TRAJECTORY OF GLIOBLASTOMA EVOLUTION
Authors: Park Jong Bae , Iavarone Antonio
Published: November 2023

The evolutionary trajectory of glioblastoma after therapy is a multifaceted biological process that extends beyond discrete genetic alterations alone. Here, we performed a proteogenomic analysis of 123 longitudinal glioblastoma pairs, temporally separated by standard-of-care treatment. Integrative analyses identified a prevalence of a highly proliferative cellular state at diagnosis and its replacement by recurrent tumors sharing activation of neuronal differentiation and synaptogenicpathways. Proteomic and phosphoproteomic analyses revealed that the molecular transition from the proliferative to the neuronal state at recurrence is marked by coherent post-translational activation of the WNT/PCP signaling pathway and the BRAF protein kinase. Multi-omic analysis of the Patient-Derived Xenograft (PDX) model demonstrated similar patterns of evolutionary trajectory, marked by activation of neuronal signaling programs that were consistently observed in longitudinal patients. Inhibition of the BRAF kinase with small molecule inhibitors impaired both neuronal differentiation and migration capability of recurrent glioblastoma cells, which are the phenotypic hallmarks of glioblastoma progression after therapy. This work provides comprehensive insights into the biological mechanisms of glioblastoma evolution and treatment resistance and highlights new therapeutic opportunities to effectively counter them in the clinic.
CNSC-09. CHI3L1 IS A MOLECULAR MARKER FOR GLIOBLASTOMA NETWORK CONNECTIVITY AND FUNCTIONALLY ORCHESTRATES TUMOR MICROTUBE FORMATION
Authors: Hoffmann Dirk , Hai Ling , Wagener Robin , Hausmann David , Azorín Daniel Domínguez , Xie Ruifan , Huppertz Magnus-Carsten , Hiblot Julien , Sievers Philipp , Ito Jakob , Weil Sophie , Cebulla Gina , Mandelbaum Henriette , Jung Erik , Kourtesakis Alexandros , Ernst Kati , Reibold Denise , Warnken Uwe , Venkataramani Varun , Will Rainer , Suva Mario , Herold-Mende Christel , Sahm Felix , Winkler Frank , Schlesner Matthias , Wick Wolfgang , Kessler Tobias
Published: November 2023

Chitinase 3-like 1 (CHI3L1) is a secreted glycoprotein and its RNA expression elevated in glioblastoma (GB) compared to other tumor types and related normal tissues. Furthermore, transcript levels in GB dictate aggressiveness through modulating stemness, proliferation and tumor microenvironment. This ultimately influences patient survival. We here provide evidence that the pathogenic relevance of CHI3L1 expression is associated with the extent of tumor microtubes (TMs) - ultralong membrane tubes that connect GB cells (GBCs) to a network with considerable relevance for tumor progression and therapy resistance. Single cell RNA profiling of xenografted GBCs with different degrees of morphological and functional TMs identified CHI3L1 as a prognostic marker for TM network extent. We demonstrate that both RNA and protein expression levels are suitable markers in preclinical in vitro systems modeling TM connectivity as well as in clinical specimens. Genetic perturbation of CHI3L1 influenced GBC network integration, caused a shift of the dominant cell state and altered the phosphorylation status of the TM-driver GAP43. Pharmacological blocking of CHI3L1 with an antibody reduced TM networks, thus providing a handle for future clinical translation. Together, these data identify a functional and upstream role of CHI3L1 in governing tumor cell connectivity, CHI3L1 RNA and protein expression as a novel way to determine overall GBC connectivity for future trials, and finally a new therapeutic target for tumor network-disrupting strategies.
CNSC-10. GLIOBLASTOMA CELLS IMITATE NEURON’S EXCITABILITY IN ACUTE AND ORGANOTYPIC HUMAN BRAIN SLICES
Authors: Tong Tong , Ozsvar Attila , Hou Wen-Hsien , Korshøj Anders
Published: November 2023

Glioblastoma (GBM) is a highly lethal cancer due to its inter- and intra-patient molecular and physiological heterogeneity, and its complex interaction with brain tissue. Recent findings demonstrate that Neuron-to-brain tumor-synaptic-communications (NBTSCs) engage in a vicious cycle of enhanced neuronal activity, driving glioma progression and invasion and tumor-induced epilepsy. However, the physiology of these synaptic interactions in the tumor-infiltrated human neocortex remains largely uncharacterized. To address this gap, our study focused on investigating the intrinsic electrical properties of glioma cells and NBTSCs in the tumor-infiltrated human cortex using a unique experimental workflow involving acute and cultured human tumor-infiltrated brain slices. Via an adeno-associated virus-assisted gene delivery technique, we selectively express fluorescence proteins in a cell-type specific manner. By performing fluorescence-guided whole-cell patch clamp recording, we characterized the electrophysiological properties of glioma cells and neurons under the tumor microenvironment. We observed that glioma cells from tumor-infiltrated human brain samples displayed depolarized resting membrane potential (-32.21 ± 2.1mV) and high input resistance (1.24 ± 0.18 GΩ). Approximately 69.2% of glioma cells exhibited spikelets or distorted-action potentials upon current injection. Consistent with other studies, a subset of glioma cells (11.8%) exhibited spontaneous excitatory postsynaptic currents (sEPSC). In addition, the tumor-infiltrated microenvironment significantly influences the proximal neurons’ physiology and the neuronal microcircuitry, leading to abnormal intrinsic electrical properties, depolarized resting membrane potential (-37.66 ± 4.32 mV), and a significant reduction in spontaneous synaptic input frequency. Collectively, our study consolidates the existence of NBTSCs in human cancer patients and reveals that glioma cells exhibit neuron-like excitability, which is contrary to the common understanding that they are considered as non-excitable. Under the tumor microenvironment, neurons display hyperexcitability. Our newly established organotypic human brain slice culture platform provides potential grounds for exploring further the biological characteristics of gliomas and NBTSCs.
CNSC-16. MOLECULAR PROFILING AND ACTIONABLE MUTATIONS IN ADULT PATIENTS WITH IDH WILD TYPE GLIOMAS AND GLIONEURONAL AND NEURONAL TUMORS
Authors: Pellerino Alessia , Marchesani Elena Maria , Bertero Luca , Ricci Alessia Andrea , Mangherini Luca , Bruno Francesco , Soffietti Riccardo , Rudà Roberta
Published: November 2023

BACKGROUND

Next generation sequencing (NGS) assays enable the identification of genetic alterations that may guide a personalized treatment in glioma patients. Material and

METHODS

From July 2021 to May 2023 a NGS analysis (Myriapod® NGS Cancer DNA and RNA panels) was performed in a cohort of 211 adults IDH wild type (wt) gliomas or circumscribed astrocytoma or glioneuronal/neuronal tumors. The aim was to identify actionable molecular alterations and provide targeted therapy at recurrence accordingly.

RESULTS

NGS analysis was available in 211 patients (95.5%), while NGS was not feasible in 10 patients (4.5%). 188/211 (89.1%) had an IDH wt glioblastoma (GBM) (median age 61 years), 6 (2.8%) had IDH wt lower grade gliomas (LGG) (median age 63 years), and 17 (8.1%) had circumscribed astrocytoma or glioneuronal/neuronal tumor (median age 37 years). Overall, actionable molecular alterations were found in 13/211 patients (6.2%). 7/211 patients (3.3%) had BRAF V600E mutation (3 IDH wt GBM, 1 IDH wt LGG, and 3 circumscribed/glioneuronal and neuronal tumor), 4/211 patients (1.9%) had FGFR3-TACC3 fusion (all GBM), 1/211 patients (0.5%) had PIK3CA mutation (GBM), and 1/211 patients (0.5%) had NTRK1 fusion (glioneuronal tumor). Other molecular mutations, including ALK, ERBB2, MET, KIT, KRAS, PDGFRA, and RET have not been detected. Three patients recurred after first-line therapy: 1 with BRAF mutated GBM, 1 with GBM harboring FGFR3-TACC3 fusion, and 1 with NTRK1 fusion positive glioneuronal tumor received specific BRAF/MEK inhibitor (dabrafenib/trametinib), FGFR inhibitor (pemigatinib), and NTRK inhibitor (larotrectinib), respectively. A remarkable activity of BRAF/MEK and NTRK inhibitors was observed (9 and 6 months, respectively *), while FGFR inhibitor did not display an activity to control disease (9 weeks). * ongoing

CONCLUSION

The incidence of actionable molecular alterations in IDH wt gliomas and glioneuronal/neuronal tumors was low. However, the use of targeted therapies may lead to significant impact on the outcome.