Methylguanine Methyltransferase Research Articles

AB059. Molecular signatures for survival prediction in glioma: a prospective, real-world data analysis.

Glioma characterization and follow-up are underreported from low-and-middle-income country centers within the literature. With the recent emphasis on molecular markers for survival prediction, there is a need for robust data exploring molecular epidemiology in these countries. In Pakistan particularly, there is a significant gap in glioma outcomes reporting and survival analysis. One hundred and sixty-five consecutive glioma patients were enrolled from 2019 onwards; histopathological and molecular analysis was performed on archived formalin-fixed paraffin-embedded (FFPE) blocks for isocitrate dehydrogenase (IDH), P53, α-thalassemia retardation X-linked (ATRX) and Ki-67 immunohistochemical (IHC) markers. Survival analysis was calculated using the Kaplan-Meier method; hazard ratios are reported through a multivariate Cox regression model. Fifty-seven (35%) histopathological diagnoses were revised according to the updated criteria; 30% (n=16) glioblastoma were converted to a new category on re-analysis. IDH wild type (IDH-WT) gliomas had a significantly worse overall survival (log-rank =0.002), with a 2-year survival rate of 60% for IDH-mutant (IDH-M) and 38% for IDH-WT. Significant survival differences were seen for the Ki-67 index (log-rank =0.001) and methylguanine methyltransferase (MGMT) promotor methylation [log-rank =0.027, 2-year survival rate: 100% (methylation detected), 33% (methylation not detected)]. On Cox proportional hazards regression, gross total resection (P<0.001), IDH mutation (P<0.001), and updated histopathological diagnosis (P<0.001) were significant predictors of survival, with good sensitivity and specificity as seen on receiver operating characteristic (ROC) analysis [area under the curve (AUC) =0.86]. In our cohort, the revised World Health Organization (WHO) classification shows significant implications on prognosis and implications for treatment. Although these markers are not commonly used in low-and-middle-income country centers, our results strongly support their greater implementation for improved prognostication and reclassification.

Dummy run for planning of isotoxic dose-escalated radiation therapy for glioblastoma used in the PRIDE trial (NOA-28; ARO-2024-01; AG-NRO-06)

BackgroundThe PRIDE trial (NOA-28; ARO-2024-01; AG-NRO-06; NCT05871021) is designed to determine whether a dose escalation with 75.0 Gy in 30 fractions can enhance the median overall survival (OS) in patients with methylguanine methyltransferase (MGMT) promotor unmethylated glioblastoma compared to historical median OS rates, while being isotoxic to historical cohorts through the addition of concurrent bevacizumab (BEV). To ensure protocol-compliant irradiation planning with all study centers, a dummy run was planned and the plan quality was evaluated. MethodsA suitable patient case was selected and the computed tomography (CT), magnetic resonance imaging (MRI) and O-(2-[18F]fluoroethyl)-L-tyrosine (FET) positron emission tomography (PET) contours were made available. Participants at the various intended study sites performed radiation planning according to the PRIDE clinical trial protocol. The treatment plans and dose grids were uploaded as Digital Imaging and Communications in Medicine (DICOM) files to a cloud-based platform. Plan quality and protocol adherence were analyzed using a standardized checklist, scorecards and indices such as Dice Score (DSC) and Hausdorff Distance (HD). ResultsMedian DSC was 0.89, 0.90, 0.88 for PTV60, PTV60ex (planning target volume receiving 60.0 Gy for the standard and the experimental plan, respectively) and PTV75 (PTV receiving 75.0 Gy in the experimental plan), respectively. Median HD values were 17.0 mm, 13.9 mm and 12.1 mm, respectively. These differences were also evident in the volumes: The PTV60 had a volume range of 219.1–391.3 cc (median: 261.9 cc) for the standard plans, while the PTV75 volumes for the experimental plans ranged from 71.5–142.7 cc (median: 92.3 cc). The structures with the largest deviations in Dice score were the pituitary gland (median 0.37, range 0.00–0.69) and the right lacrimal gland (median 0.59, range 0.42–0.78). ConclusionsThe deviations revealed the necessity of systematic trainings with appropriate feedback before the start of clinical trials in radiation oncology and the constant monitoring of protocol compliance throw-out the study. Trial registrationNCT05871021

Abstract 992: The impact of tamoxifen on autophagic and epigenetic regulation in endometrial cancer

Abstract Background: Endometrial cancer, predominantly endometrioid adenocarcinoma, ranks as the fourth most common cancer in women globally, with an increasing incidence over the last two decades. Tamoxifen, well-known for its antiestrogenic characteristics in the treatment of breast cancer, paradoxically increases the risk of endometrial cancer. Understanding this dual role of tamoxifen is crucial for optimizing breast cancer therapy while mitigating endometrial cancer risk. This study aims to elucidate the molecular alterations induced by tamoxifen in endometrial thickening and its potential to induce cancer, particularly focusing on autophagy, epithelial-mesenchymal transition (EMT), and cancer stem-like cells (CSCs). Methods: In this study, female BALB/c mice were administered tamoxifen (20 mg/ml) orally for five consecutive days. We monitored changes in EMT markers, autophagy-related genes, and stem cell markers over 1 to 5 months. Furthermore, we investigated the epigenetic modulation of key genes involved in these processes. Results: Tamoxifen treatment led to altered expression of both epithelial and mesenchymal EMT markers, autophagy-related genes (p62, LC3B, Beclin-1, mTOR, ATG12, ATG16), and stem cell markers (Nanog, SOX2, ALDH1). Notably, we observed significant epigenetic changes, including increased methylation of E-cadherin and decreased methylation of Twist. Furthermore, we observed hypermethylation (a process often linked to the silencing of tumor-suppressing genes) in MGMT (Methylguanine methyltransferase), a DNA repair enzyme involved in chemoresistance, and DAPK (Death-associated protein kinase), a kinase associated with apoptosis, autophagy, and inflammation. We also observed hypermethylation in the CpG promoter regions of autophagy-related genes (Beclin-1 and LC3B) highlighting a potential mechanism for tamoxifen-induced tumorigenic changes. Conclusion: Our findings demonstrate tamoxifen's influence on cellular and molecular pathways, highlighting the complex interplay between drug treatment and cancer progression. The observed epigenetic changes provide insight into the mechanisms by which tamoxifen may contribute to endometrial cancer risk, suggesting potential targets for therapeutic intervention. Citation Format: Shilpa Thota, Rizwana Begum, Naveen Chintala, Abhishek Pandit, Biplov Sapkota, Joseph Francis. The impact of tamoxifen on autophagic and epigenetic regulation in endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 992.

DNAR-04. MICRORNA DEGRADATION ENHANCES GLIOBLASTOMA INTRA-TUMORAL HETEROGENEITY TO AUGMENT ACQUIRED TEMOZOLOMIDE RESISTANCE

Abstract INTRODUCTION O6 methyl-guanine methyltransferase (MGMT) restores alkylated DNA to its undamaged form and dictates clonal evolution in response to chemotherapy. As such, MGMT is a major determinant of glioblastoma resistance to the standard-of-care chemotherapy temozolomide. miR-181d is a MGMT suppressing microRNA, whose expression inversely correlate with survival expectation after temozolomide treatment. Here, we characterize the expression of miR-181d and MGMT in response to temozolomide treatment. Method: Laboratory characterization, single cell analysis, and murine modeling RESULTS Treatment of glioblastoma cells with temozolomide (TMZ) induced a feed-forward cascade resulting in an ataxia telangiectasia and Rad3 (ATR) and polyribonucleotide nucleotidyl transferase 1 (PNPT1) dependent degradation of miR-181d. Single-cell analyses revealed such miR-181d degradation: 1) increased mean level of MGMT expression in a cell population and 2) enhanced cell-to-cell variability in MGMT expression to increase intra-tumoral heterogeneity in DNA repair capacity. This increased intra-tumoral heterogeneity enhanced glioblastoma resistance to temozolomide through a mechanism distinct from lowering the mean MGMT expression of the tumor population. Suppression of this heterogeneity by exogenous miR-181d over-expression enhanced the tumoricidal activity of temozolomide against glioblastomas. CONCLUSION We identified an interface between DNA damage response and miRNA degradation that mediate intra-tumoral heterogeneity and subsequent temozolomide resistance. This resistance mechanism is distinct from modulation of the mean MGMT expression of the population.

NCOG-36. IMPACT OF COLLAGEN TILE BRACHYTHERAPY AS TREATMENT FOR RECURRENT GLIOBLASTOMAS ON FUNCTIONAL STATUS AND QUALITY OF LIFE

Abstract BACKGROUND Recurrent glioblastoma is an aggressive disease with dismal prognosis despite advances in standard therapy, making the maintenance of functional status and quality of life (QOL) in patients an important endpoint during treatment. Here, we report functional status (KPS) and QOL metrics (LASA and FACT-Br) at 6-months post-treatment for recurrent glioblastoma patients treated with maximal safe resection followed by intraoperative placement of collagen tile brachytherapy with Cesium-131 (Surgically Targeted Radiation Therapy or STaRT), a novel brachytherapy carrier. OBJECTIVE To assess the impact of STaRT on KPS and QOL among patients with recurrent glioblastoma (rGBM) at 6-months post-treatment. METHODS Patients were treated between 10/2020 and 05/2023 as part of a multi-institutional registry study (NCT#04427384). KPS was used to measure functional status. Linear Analog Self-Assessment (LASA) and Functional Assessment of Cancer Therapy – Brain (FACT-Br) were used to measure QOL. All assessments were collected at pre-surgery, and 1,3, and 6-months post-treatment. RESULTS 57 rGBM patients were treated on the Registry between 10/2020 and 05/2023. Of the 57 participants, 47 participants remain in the study, with 21 participants reaching the 6-month time point (5 exits, 5 deaths). Median age was 60 years (range 28-81). Methylguanine methyltransferase (MGMT) promoter was methylated in 17.5%, unmethylated in 38.5%, and unknown in 44%. Gross total resection was achieved in 72% of participants. Median KPS was 80% (range 40%-100%) at screening versus 70% (range 40%-90%) at 6 months post-treatment. Median LASA was 37 (range 8-50) at pre-surgery versus 34 (range 5-48) at 6-months post-treatment. Minimally important differences (MID) were noted between pre-surgery (136.9 ± 29.5) and 6-months post-treatment (112.5 ± 35.7) for the FACT-Br Total Score. CONCLUSION This interim data analysis supports further investigation of STaRT as a treatment for recurrent glioblastomas as a means of providing stable functional status and quality of life.

Significance of O6-methyl guanine methyltransferase promoter methylation in high grade glioma patients: optimal cutoff point, CpG locus, and genetic assay.

To evaluate O6-methyl guanine methyltransferase (MGMT) promoter methylation status in high grade glioma patients and to identify the best cutoff point as well as the most predictive CpG loci for patients survival. Consecutive high grade glioma patients treated with surgical gross total resection followed by concomitant radiochemotherapy and adjuvant chemotherapy were included in this retrospective observational study. Methylation status of MGMT promoter CpG island of resected tumor tissue were evaluated using next generation sequencing assay. The outcomes were grouped as CpG 70-78, CpG 79-83, CpG 84-87, CpG 70-87, and whole promoter. Quantitative analyses were dichotomized as methylated or unmethylated based on the cutoff points set to %10, and methylation was further graded as <%10 unmethylated, %10-30 low-methylated, and %30-100 high-methylated. Total of 95 patients with the mean age of 51.50 ± 12.36 years were included in the study. Overall survival (OS) and progression free survival (PFS) were 14.53 ± 1.92 (95% CI 10.77-18.30) and 10.90 ± 2.05 (95% CI 6.89-14.92) months, respectively. MGMT promoter was methylated in 38.2% of cases and high-methylated in 10.5% of cases. Methylation status of MGMT promoter was recognized as a very powerful predictor of OS and PFS. In particular, high-methylation of CpG 79-83 and CpG 84-87 islands at promoter region were strongly associated with better survival outcomes (p < 0.05). Our outcomes support the prognostic value of MGMT promoter methylation in patients with high grade glioma. Sequencing of whole promoter CpG islands demonstrated that methylation of particular CpG sites might predict clinical outcomes more precisely.

Temozolomide-fatty acid conjugates for glioblastoma multiforme: In vitro and in vivo evaluation.

Glioblastoma multiforme (GBM) is the deadliest brain tumor with a poor prognosis and limited therapeutic options. Temozolomide (TMZ) is the first-line chemotherapeutic agent used for the treatment of GBM; however, it suffers from several limitations, including short half-life, rapid metabolism, <1% brain bioavailability, methyl guanine methyl transferase (MGMT) based chemoresistance, and hematological toxicities. Several approaches have been adopted to overcome these limitations, particularly by using nanotechnology-based systems, but its physicochemical properties make TMZ challenging to load into these nanocarriers. In the current research, we conjugated TMZ with different fatty acids, i.e., linoleic acid (LA), oleic acid (OA), and palmitic acid (PA), to obtain TMZ-fatty acid conjugates, which are comparatively hydrophobic, less prone to degradation and potent. These conjugates were thoroughly characterized using 1H NMR spectroscopy, high-resolution mass spectrometry (HR-MS), and reverse phase-high performance liquid chromatography (RP-HPLC). The synthesized conjugates, namely Temozolomide-oleic acid (TOA,6R1), Temozolomide-linoleic acid (TLA, 6R2), and Temozolomide-palmitic acid (TPA, 6R3), showed an IC50 of 101.4, 67.97, and 672.04μM, respectively in C6 cells and 428.257, 366.43 and 413.69μM, respectively in U87-MG cells. On the other hand, the free TMZ showed an IC50 of >1000μM and 564.23μM in C6 and U87-MG, respectively. Further, the in vivo efficacy of the TMZ-fatty acid conjugates was evaluated in the C6-induced orthotropic rat glioblastoma model, wherein the TMZ-fatty acid conjugate showed improved survival rate (1.6 folds) and overall health of the animals. Collectively, the conjugation of fatty acids with TMZ improves its anticancer potential against glioblastoma multiforme (GBM).

O6 -methylguanine methyltransferase promoter methylation status of glioblastoma cell line clonal population.

Glioblastoma (GBM) remains a treatment-resistant malignant brain tumor in large part because of its genetic heterogeneity and epigenetic plasticity. In this study, we investigated the epigenetic heterogeneity of GBM by evaluating the methylation status of the O6 -methylguanine methyltransferase (MGMT) promoter in individual clones of a single cell derived from GBM cell lines. The U251 and U373 GBM cell lines, from the Brain Tumour Research Centre of the Montreal Neurological Institute, were used for the experiments. To evaluate the methylation status of the MGMT promoter, pyrosequencing and methylation-specific PCR (MSP) were used. Moreover, mRNA and protein expression levels of MGMT in the individual GBM clones were evaluated. The HeLa cell line, which hyper-expresses MGMT, was used as control. A total of 12 U251 and 12 U373 clones were isolated. The methylation status of 83 of 97 CpG sites in the MGMT promoter were evaluated by pyrosequencing, and 11 methylated CpG sites and 13 unmethylated CpG sites were evaluated by MSP. The methylation status by pyrosequencing was relatively high at CpG sites 3-8, 20-35, and 7-83, in both the U251 and U373 clones. Neither MGMT mRNA nor protein was detected in any clone. These findings demonstrate tumor heterogeneity among individual clones derived from a single GBM cell. MGMT expression may be regulated, not only by methylation of the MGMT promoter but by other factors as well. Further studies are needed to clarify the mechanisms underlying the epigenetic heterogeneity and plasticity of GBM.

Cognitive functioning in a cohort of high-grade glioma patients.

High grade gliomas are associated with cognitive problems. The aim of the study was to investigate cognitive functioning in a cohort of patients with high grade glioma, according to isocitrate dehydrogenase (IDH) and methyl guanine methyl transferase (MGMT) status and other clinical characteristics. The patients with the high-grade glioma treated in Slovenia in given period of time were included in study. Postoperatively they completed neuropsychological assessment consisting of Slovenian Verbal Learning Test, Slovenian Controlled Oral Word Association Test, Trail Making Test Part A and B and self-evaluation questionnaire. We analysed results (z-scores and dichotomized results) also according to IDH mutation and MGMT methylation. We examined differences between groups using T-test, Mann-Whitney U, χ2 and Kendall's Tau tests. Out of 275 patients in the cohort, we included 90. Forty-six percent of patients were unable to participate due to poor performance status and other conditions related to tumour. Patients with the IDH mutation were younger, with better performance status, larger proportions of grade III tumours and MGMT methylation. In this group cognitive functioning is significantly better in the domains of immediate recall, short delayed recall and delayed recall, and in the fields of executive functioning and recognition. There were no differences in cognitive functioning in regard to MGMT status. Grade III tumours were associated with more frequent MGMT methylation. Self-assessment proved week tool, associated only with immediate recall. We found no differences in cognitive functioning according to MGMT status, but cognition was better when IDH mutation was present. In a cohort study of patients with high-grade glioma, almost half were unable to participate in a study, which points to an overrepresentation of patients with better cognitive functioning in the research.

865 Survival Benefit of Stereotactic Laser Ablation (SLA) in Newly Diagnosed Glioblastoma Patients: A Matched Cohort Analysis

INTRODUCTION: Post-hoc analysis of a previously published matched cohort study suggests improved survival for glioblastoma patients who underwent stereotactic laser ablation (SLA) relative those who underwent a biopsy, particularly for tumors where contrast enhancing (CE) regions were completely covered by thermal ablation. METHODS: Thirteen patients with newly diagnosed isocitrate dehydrogenase (IDH) wild type glioblastomas (without H3K27 mutation) who underwent SLA with ablation coverage of &gt;90% of the contrast enhancing region were identified. Control cohort was generated by screening patients who underwent stereotactic biopsy for a newly diagnosed tumor matched for: age (+/- 10 years), location, tumor volume (+/- 30% of CE volume), and methyl-guanine methyl-transferase (MGMT promoter methylation status). Post-operative course, complications, 30-day readmission, and overall survival data were collected. RESULTS: The mean KPS for the SLA and biopsy cohorts were comparable (SLA: 90+14; biopsy: 87+16). One patient (8%) in the SLA cohort experienced new-onset diplopia, and one biopsy-only patient (8%) suffered hemi-sensory alteration. There were no 30-day readmissions for either cohort. With a median follow-up of 283 days, the median survival (mOS) for 375 days (∼13 mo) for the SLA treated patient cohort and 264 days (∼ 9 mo) for the biopsy cohort (p = 0.026). To exclude the possibility that this survival difference was due to selection of prognostically favorable patients for SLA, the survival benefit was confirmed by comparison to a second matched biopsy-only glioblastoma cohort from another institution without capacity for SLA. When stratified by MGMT promoter methylation, SLA survival benefit was significant in only patients with methylated tumors (SLA mOS: 620 days (21 mo); biopsy mOS: 171 days (5.7 mo), p = 0.007) CONCLUSIONS: For MGMT promoter methylated glioblastomas, SLA treatment was associated with improved survival relative to biopsy-treated patients. Validation of this finding in a prospective study is warranted.

The Prognostic Impact of Gender, Therapeutic Strategies, Molecular Background, and Tumor-Infiltrating Lymphocytes in Glioblastoma: A Still Unsolved Jigsaw.

Despite the adoption of novel therapeutical approaches, the outcomes for glioblastoma (GBM) patients remain poor. In the present study, we investigated the prognostic impact of several clinico-pathological and molecular features as well as the role of the cellular immune response in a series of 59 GBM. CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) were digitally assessed on tissue microarray cores and their prognostic role was investigated. Moreover, the impact of other clinico-pathological features was evaluated. The number of CD4+ and CD8+ is higher in GBM tissue compared to normal brain tissue (p < 0.0001 and p = 0.0005 respectively). A positive correlation between CD4+ and CD8+ in GBM is present (rs = 0.417-p = 0.001). CD4+ TILs are inversely related to overall survival (OS) (HR = 1.79, 95% CI 1.1-3.1, p = 0.035). The presence of low CD4+ TILs combined with low CD8+ TILs is an independent predictor of longer OS (HR 0.38, 95% CI 0.18-0.79, p = 0.014). Female sex is independently related to longer OS (HR 0.42, 95% CI 0.22-0.77, p = 0.006). Adjuvant treatment, methylguanine methyltransferase (MGMT) promoter methylation, and age remain important prognostic factors but are influenced by other features. Adaptive cell-mediated immunity can affect the outcomes of GBM patients. Further studies are needed to elucidate the commitment of the CD4+ cells and the effects of different TILs subpopulations in GBM.

Multiparametric Magnetic Resonance Imaging Correlates of Isocitrate Dehydrogenase Mutation in WHO high-Grade Astrocytomas.

Isocitrate dehydrogenase (IDH) mutation and O-6 methyl guanine methyl transferase (MGMT) methylation are surrogate biomarkers of improved survival in gliomas. This study aims at studying the ability of semantic magnetic resonance imaging (MRI) features to predict the IDH mutation status confirmed by the gold standard molecular tests. The MRI of 148 patients were reviewed for various imaging parameters based on the Visually AcceSAble Rembrandt Images (VASARI) study. Their IDH status was determined using immunohistochemistry (IHC). Fisher's exact or chi-square tests for univariate and logistic regression for multivariate analysis were used. Parameters such as mild and patchy enhancement, minimal edema, necrosis &lt; 25%, presence of cysts, and less rCBV (relative cerebral blood volume) correlated with IDH mutation. The median age of IDH-mutant and IDH-wild patients were 34 years (IQR: 29-43) and 52 years (IQR: 45-59), respectively. Mild to moderate enhancement was observed in 15/19 IDH-mutant patients (79%), while 99/129 IDH-wildtype (77%) had severe enhancement (p-value &lt;0.001). The volume of edema with respect to tumor volume distinguished IDH-mutants from wild phenotypes (peritumoral edema volume &lt; tumor volume was associated with higher IDH-mutant phenotypes; p-value &lt; 0.025). IDH-mutant patients had a median rCBV value of 1.8 (IQR: 1.4-2.0), while for IDH-wild phenotypes, it was 2.6 (IQR: 1.9-3.5) {p-value = 0.001}. On multivariate analysis, a cut-off of 25% necrosis was able to differentiate IDH-mutant from IDH-wildtype (p-value &lt; 0.001), and a cut-off rCBV of 2.0 could differentiate IDH-mutant from IDH-wild phenotypes (p-value &lt; 0.007). Semantic imaging features could reliably predict the IDH mutation status in high-grade gliomas. Presurgical prediction of IDH mutation status could help the treating oncologist to tailor the adjuvant therapy or use novel IDH inhibitors.

RBIO-03. A MULTI-INSTITUTIONAL EXPERIENCE OF GAMMATILE BRACHYTHERAPY AS TREATMENT FOR RECURRENT GLIOBLASTOMAS

Abstract BACKGROUND Recurrent glioblastoma remains a disease without established standard of care. Here, we report the safety profile and survival outcome of recurrent glioblastoma patients treated with maximal safe resection followed by intraoperative placement of GammaTile (GT), a collagen tile-embedded Cs-131 brachytherapy platform. METHODS The study included isocitrate Dehydrogenase (IDH) wild-type glioblastoma patients who suffered tissue confirmed recurrence after concurrent temozolomide-radiation therapy. The series included consecutively patients treated at the University of Minnesota, Barrow Neurological Institute, Vidant Health, and Emory University Hospital. Survival results were stratified by methyl-guanine methyl transferase (MGMT) promoter methylation status RESULTS In total 48 patients were treated between 2019 and 2022 in this multi-institutional experience. The median days of hospitalization following the procedure was two (range: 1-15), with 72.5% of the GT implanted patients discharged home before postoperative day three. In terms of post-operative course, two patients suffered new-onset seizure post-surgery (4.0%). There was one (2%) 30-day mortality from intracranial hemorrhage secondary to heparinization for an ischemic limb. There were 13% readmission within 30 days, including hydrocephalus (n=2), urinary tract infection (n=1), deep venous thrombosis (n=1), failure to thrive (n=1), and infected wound (n=1). Of note, the patient with the infected wound had suffered a previous wound infection prior to the GT implantation and the pathogenic organism for both infections were the same (S. aureus). With a median follow-up of 250 days, twelve-month local control was 82%. Median overall survival (OS) from the time of diagnosis was 19 and 33 months for the MGMT unmethylated and methylated tumors, respectively. Select patients (n = 6) treated with adjuvant ketogenic diet in addition to GT and subsequent chemotherapy exhibited survival beyond expectation (&amp;gt; 14 mo, median survival not reached). CONCLUSION This multi-institutional experience supports further investigation of GT brachytherapy as a treatment for recurrent glioblastomas.

EPID-24. TIMING OF STANDARD-OF-CARE CHEMO-RADIATION THERAPY FOLLOWING SURGICAL RESECTION OR STEREOTACTIC BIOPSY

Abstract OBJECT The literature examining the optimal timing for initiating post-surgical therapy after glioblastoma surgery is brimming with inconsistencies, with reports of both deleterious effects and benefits in the delay of post-surgical therapy at various time intervals. Moreover, there is currently limited data pertinent to the post-Stupp regimen era. Here, we use the National Cancer Database (NCDB) to explore this matter, with a focus on patients who underwent the standard-of-care (SOC) Stupp regimen. METHODS The study cohort included newly glioblastoma patients from the NCDB, with exclusion of patients age &amp;lt; 40 [as proxy for isocitrate dehydrogenase (IDH) mutated astrocytoma] and stratified for Methyl-guanine-methyl transferase (MGMT) promotor methylation. Timing of initiating SOC was grouped by 0-2, 2-4, 4-6, 6-8, and &amp;gt; 8 weeks from surgery. Biopsy and craniotomy patients were analyzed separately. The Kaplan-Meier method and Cox proportional hazards regression were applied for survival analysis. RESULTS We identified 6511 glioblastoma patients who underwent SOC therapy with available MGMT methylation status. SOC was initiated in 228 (3.5%), 2478 (38.1%), 2954 (45.4%), 646 (9.9%), and 205 (3.2%) patients at 0-2, 2-4, 4-6, 6-8, and &amp;gt;8 weeks after surgery. For craniotomy patients, initiation of SOC at 0-2 weeks was associated with an increased risk of death relative to SOC initiation at 4-6 weeks (HR: 1.19, p = 0.036). These results remain robust after controlling for the extent of resection, tumor size, and tumor location (HR: 1.19, p = 0.044). Such association was not observed in patients who underwent stereotactic biopsy (HR: 1.27, p = 0.148). Patients who received SOC at 6-8 weeks post-craniotomy (HR: 0.96, p = 0.420) or biopsy (HR: 0.93, p = 0.628) did not appear to worsen survival outcomes. CONCLUSION Optimal timing for initiation of SOC differ for craniotomy and needle biopsy patients. Initiating SOC within 2 weeks after craniotomy could lead to worse survival outcomes for GBM patients.

EPID-21. INCORPORATING RECEIPT OF STANDARD-OF-CARE AS A KEY VARIABLE IN POPULATION-BASED STUDY OF GLIOBLASTOMA SURVIVAL

Abstract Object Populational database research with real-world settings offers epidemiologic insight at scales that cannot be easily recapitulated, especially for rare cancers such as glioblastoma. However, most survival studies were conducted without consideration for whether the patients received the standard of care (SOC). METHODS Survival analysis was performed using the National Cancer Database (NCDB) and stratified for Methyl-guanine-methyl transferase (MGMT) promotor methylation. Glioblastoma patients age &amp;lt; 40 were excluded to minimize the prognostic impact of isocitrate dehydrogenase (IDH) mutations. SOC was defined as the receipt of chemotherapy and radiation within 2-6 weeks of surgical biopsy or resection. Sensitivity analyses were performed allowing chemotherapy and radiation start date to differ by 0, 3, 5, and 10 days. RESULTS Within the NCDB (N = 11231), 54.7% of newly diagnosed glioblastoma patients did not receive SOC. The median survival time (MST) estimates were significantly shorter for all glioblastoma patients (10.9, IQR: 4.3-21.5, months) relative to SOC treated patients (15.3, IQR: 8.6-26.2, months; p &amp;lt; 0.001). The MST estimates remain stable in sensitivity analysis allowing for 0, 3, 5, and 10-day discrepancy between chemotherapy and radiation start date. These results were recapitulated using the SEER-Medicare database. The MST estimates for MGMT methylated and unmethylated glioblastoma patients after SOC in the NCDB were 21.0 and 14.5-mo, respectively. The overall and MGMT-specific survival estimates mirror those reported by the SOC arms of five recent randomized controlled studies. Analysis of all glioblastoma patients indicates survival benefit of immunotherapy (HR: 0.90, p = 0.020) while such benefit was absent in SOC treated patients (HR: 0.97, p = 0.644) - with the latter observation more reflective with trial results and clinical experiences. CONCLUSION Survival analysis in the populational database study can be misleading without information on the receipt of SOC.

TMET-08. METABOLOMIC CHANGES IN GLIOBLASTOMA PATIENTS UNDERGOING CONCURRENT CHEMORADIATION THERAPY

Abstract OBJECTIVE To illustrate changes of untargeted metabolomics in patients with glioblastoma IDH wildtype undergoing concurrent radiation therapy (RT) with temozolomide (TMZ). This study implemented machine learning (ML) algorithms to predict treatment phase: pre-surgery, post-surgery, pre-radiation, and post radiation based on untargeted metabolomics data. METHODS Thirty-six patients with glioblastoma IDH wildtype (18 methylguanine methyltransferase [MGMT] methylated, 16 MGMT unmethylated, 2 MGMT status unknown) were enrolled into this study. Serum samples obtained from patients on the same day before surgery, 2 days after surgery, before starting their concurrent chemoradiation, and after concluding this phase of treatment. Blood samples were obtained via antecubital phlebotomy without regard for time of the day, diet, or fasting status. Untargeted metabolomics by GC-TOF mass spectrometry were obtained and compared. The proposed ML models analyzed 105 samples from 36 patients utilizing 157 structurally identified blood metabolites. Multinomial Logistic Regression (MLR) and GradientBoostingClassifier (GB Classifier) were used to classify patient samples based on detected changes in blood metabolites. The classification performance of these models was evaluated using performance metrics and AUC scores. RESULTS Post radiation; significant increase in the following metabolites: glycine, serine, threonine, oxoproline, 6-deoxyglucose, gluconic acid, glycerol-alpha-phosphate, ethanolamine, propyleneglycol, triethanolamine, xylitol, and fumaric acid were noted while significant decrease in 3-aminopiperidine 2,6-dione was noted post radiation. MLR produced 78% accuracy, 75% precision, and AUC = 0.89, and GB Classifier produced 75% accuracy, 77% precision and AUC = 0.91. Finally, we presented a pattern of metabolites changes per clinical stage based on pairwise correlations. CONCLUSIONS This study represent the first serum metabolic signature associated with RT in patients with glioblastoma. The results from the classification algorithms and pairwise correlations showed that blood metabolites have the potential to predict phase of treatment and potentially enable to evaluate response to treatment in patients with glioblastoma in a relatively small cohort.

A Novel Epigenetic Marker Predicts Treatment Response in Glioblastoma and Provides a Target for Overcoming Treatment Resistance

<h3>Purpose/Objective(s)</h3> Glioblastoma (GBM) is a highly aggressive primary brain tumor. A major challenge in GBM treatment is tumor resistance to radiation and chemotherapy. An important hallmark of GBM is the frequent mutation of epigenetic modifiers resulting in alteration of epigenetic signaling pathways in tumor cells. However, the effect of epigenetic signaling on chemotherapy response in GBM remains to be elucidated. SETD2 is a unique histone methyl transferase that facilitates H3K36 tri-methylation. Here, we unveil the role of SETD2 mutations that frequently occur in GBM in tumor resistance to temozolomide chemotherapy. <h3>Materials/Methods</h3> We have performed next-generation sequencing of the SETD2 gene and immunohistochemical staining if its product H3K36me3 in 129 GBM patient samples that was supplemented with TCGA database analysis of additional 279 GBM samples. We used primary patient-derived GBM lines to investigate the mechanism of chemotherapy resistance in SETD2-mutant GBM. <h3>Results</h3> We demonstrate that inactivating SETD2 mutations occur in up to 13% of GBM patients. Our investigation reveals that SETD2 mutations are associated with reduced progression-free (Median PFS 0.93 vs. 0.44 years, p=0.0022) and overall survival (Median survival 1.69 vs. 1.30 years, p=0.0044) in patients with methylated MGMT (methyl-guanine methyl transferase) promotor who received temozolomide suggesting that mutation of SETD2 is a predictive marker for treatment outcomes in GBM. Consequently, we demonstrate that loss of SETD2 results in reduced H3K36me3 levels in patient samples and a profound resistance to temozolomide in primary patient-derived GBM lines. MGMT-deficient tumors can acquire chemoresistance due to disrupted mismatch repair (MMR), a DNA repair pathway that plays a critical role in converting primary temozolomide-induced DNA lesions into toxic DNA double-strand breaks. Strikingly, we found that SETD2 loss abrogates the expression of the MMR factor MSH6 indicating that chemoresistance in SETD2-deficient cells us due to disrupted MMR. Mechanistically, we show that SETD2 regulates MMR by promoting transcription of the <i>MSH6</i> gene in GBM. Epigenetic modifiers have specific antagonists capable of reversing chromatin alterations induced by these modifiers. This provides a unique opportunity to restore chemotherapy response in SETD2-mutant GBM by targeting the antagonists of SETD2. We demonstrate that combined targeting of H3K36me3-specific histone de-methylases KDM4A and NO66 restores H3K36me3 levels along with MSH6 expression and sensitivity to temozolomide in SETD2-deficient GBM cells. <h3>Conclusion</h3> Our findings establish SETD2 mutation as a novel molecular marker predictive of treatment response in GBM and provide a framework for a novel approach to overcome chemotherapy resistance in this malignant brain tumor by targeting an epigenetic signaling pathway.

Thymoquinone induces apoptosis in temozolomide-resistant glioblastoma cells via the p38 mitogen-activated protein kinase signaling pathway.

Temozolomide (TMZ) can cross the blood-brain barrier (BBB) and deliver methyl groups to the purine (guanine) bases of DNA, leading to mispairing during DNA replication and subsequent cell death. However, increased expression of the repair enzyme methyl guanine methyltransferase (MGMT), which removes methyl groups from purine bases, counteracts methylation by TMZ. We evaluated the anticancer potential of thymoquinone (TQ), a hydrophobic flavonoid that inhibits resistance and induces apoptosis in various cancer cells, both in vitro and in vivo. In vitro experiments showed that compared with the Hs683 and M059J cell lines, U251 cells were more sensitive to TMZ. Compared to U251 cells, U251R cells, a TMZ drug-resistant strain established in this study, are characterized by increased expression of phosphorylated extracellular signal-regulated kinase (p-ERK) and MGMT. TQ treatments induced apoptosis in all cell lines. The p38 mitogen-activated protein kinase signal pathway was mainly activated in U251 and U251R cells; however, p-ERK and MGMT upregulation could not suppress TQ effects. Furthermore, si-p38 pretreatment of U251R cells in TQ treatments inhibited cell apoptosis. We speculate that TQ contributed to the phosphorylation and activation of p38, but not of ERK-induced apoptosis (irrespective of TMZ resistance). In vivo, U251R-derived tumors subcutaneously inoculated in nude mice exhibited significant tumor volume reduction after TQ or TQ + TMZ cotreatments. High-performance liquid chromatography assay confirmed the presence of TQ in murine brain tissues. Our findings demonstrate that TQ can effectively cross the BBB and function alone or in combination with TMZ to treat glioblastoma.

Inhibitory effects of temozolomide on glioma cells is sensitized by RSL3-induced ferroptosis but negatively correlated with expression of ferritin heavy chain 1 and ferritin light chain

Invasive growth of glioblastoma makes residual tumor unremovable by surgery and leads to disease relapse. Temozolomide is widely used first-line chemotherapy drug to treat glioma patients, but development of temozolomide resistance is almost inevitable. Ferroptosis, an iron-dependent form of non-apoptotic cell death, is found to be related to temozolomide response of gliomas. However, whether inducing ferroptosis could affect invasive growth of glioblastoma cells and which ferroptosis-related regulators were involved in temozolomide resistance are still unclear. In this study, we treated glioblastoma cells with RSL3, a ferroptosis inducer, in vitro (cell lines) and in vivo (subcutaneous and orthotopic animal models). The treated glioblastoma cells with wild-type or mutant IDH1 were subjected to RNA sequencing for transcriptomic profiling. We then analyze data from our RNA sequencing and public TCGA glioma database to identify ferroptosis-related biomarkers for prediction of prognosis and temozolomide resistance in gliomas. Analysis of transcriptome data from RSL3-treated glioblastoma cells suggested that RSL3 could inhibit glioblastoma cell growth and suppress expression of genes involved in cell cycle. RSL3 effectively reduced mobility of glioblastoma cells through downregulation of critical genes involved in epithelial-mesenchymal transition. Moreover, RSL3 in combination with temozolomide showed suppressive efficacy on glioblastoma cell growth, providing a promising therapeutic strategy for glioblastoma treatment. Although temozolomide attenuated invasion of glioblastoma cells with mutant IDH1 more than those with wild-type IDH1, the combination of RSL3 and temozolomide similarly impaired invasive ability of glioblastoma cells in spite of IDH1 status. Finally, we noticed that both ferritin heavy chain 1 and ferritin light chain predicted unfavorable prognosis of glioma patients and were significantly correlated with mRNA levels of methylguanine methyltransferase as well as temozolomide resistance. Altogether, our study provided rationale for combination of RSL3 with temozolomide to suppress glioblastoma cells and revealed ferritin heavy chain 1 and ferritin light chain as biomarkers to predict prognosis and temozolomide resistance of glioma patients.

64-Year-Old Woman With Aphasia and Troponin Elevation

64-Year-Old Woman With Aphasia and Troponin Elevation