MGMT Methylation Research Articles

Sex differences in glioblastoma response to treatment: Impact of MGMT methylation.

Sex differences in glioblastoma response to treatment: Impact of MGMT methylation.

Unveiling the role of MGMT and DAPK hypermethylation in response to anti-EGFR agents: Molecular insights for advancing HNSCC treatment.

Epidermal growth factor receptor (EGFR) is frequently activated in head and neck squamous cell carcinoma (HNSCC) and serves as a valuable target for therapy. Despite the availability of the EGFR inhibitors Cetuximab, Afatinib, and Allitinib, there are limited predictive markers for their response. Understanding molecular aberrations in HNSCC could facilitate the identification of new strategies for patient clinical and biological classification, offering novel therapeutic avenues. We assessed CCNA1, DCC, MGMT, CDKN2A/p16, and DAPK methylation status in HNSCC cell lines and their association with anti-EGFR treatment response. MGMT methylation status displayed high sensitivity and specificity in distinguishing sensitive and resistant HNSCC cell lines to Afatinib (AUC = 0.955) and Allitinib (AUC = 0.935). Moreover, DAPK methylation status predicted response to Allitinib with high accuracy (AUC = 0.852), indicating their putative predictive biomarker roles. These findings hold promise for the development of more personalized and effective treatment approaches for HNSCC patients.

10214-COT-24 A RARE CLINICAL CASE OF LUMBAR METASTASIS FROM GLIOSARCOMA

Abstract Gliosarcoma (GS) is a variant of glioblastoma (GBM) and has been shown to be prone to extracranial metastasis. We report a rare case of lumbar metastasis of Gliosarcoma (GS) based on histopathological findings.37-year-old male.He underwent emergency surgery for a left frontal subcortical hemorrhage. Pathological diagnosis confirmed GS. Postoperatively, the patient underwent conventional radiation chemotherapy. Later, in conjunction with cranioplasty, the remaining lesion was additionally excised and maintenance therapy was administered for 9 months. Subsequently, a follow-up MRI revealed an intracranial disseminated lesion, although the primary site was not affected. At the same time, he had difficulty moving due to back pain, and MRI revealed extracranial metastases in the second lumbar vertebra and retroperitoneum. Lumbar biopsy confirmed the diagnosis of metastasis. Pathological findings at the initial surgery showed that the tumor consisted of GS and gemistocyte-like components, and immunostaining and genetic findings showed IDH wild type GBM. Additional tissue samples showed predominantly gemistocyte-like components and no GS component, and the genetic profile was similar.Lumbar biopsies showed predominantly GS and no gemistocyte-like components. MGMT methylation changed from methylation-negative to positive and then negative by immunostaining and MS-PCR. In a previous report, Feldheim et al. performed a meta-analysis of MGMT methylation changes in relapsing GBM and reported increased methylation after relapse. In addition, Jimenez et al. quantitatively analyzed MGMT methylation in recurrent GBM by pyrosequencing and found an increased rate of methylation in about half of the cases. These reports indicate that MGMT methylation is altered following therapeutic intervention. In the present case, it was hypothesized that an intracranial lesion that was initially unmethylated was methylated by the intervention, but an unmethylated, treatment-resistant GS component metastasized to the extracranial space. The clinical course of this case was a rare example of evidence of a change in MGMT methylation due to therapeutic intervention.

10098-MPC-10 HOW SHOULD WE RESPOND TO REPEATED WHO REVISIONS? IN-HOUSE MOLECULAR DIAGNOSIS SYSTEM BY ADVANCED MEDICAL SYSTEM

Abstract BACKGROUND Since the WHO2016 revision, required molecular markers has been increasing, placing a burden on clinical practice of diffuse gliomas. We established an in-house molecular diagnostic platform by Senshin-Iryo (meaning advanced medical care system) and have since been partially modifying the analysis method in accordance with the WHO2021 revision. We review our achievements in total 5 years. METHODS Analyses of IDH, BRAF, and H3 point mutations, loss of heterozygosity on 1p19q and chromosomes 10 and 17, and MGMT methylation were combined into a set and submitted as a Senshin-Iryo "Drug resistance gene testing for anticancer chemotherapy" and approved in August 2018. Subsequently, in October 2021, Sanger sequencing for TERT promotor mutation was added to the set and the LOH analysis was replaced to MLPA, in order to analyze 1p19q codeletion and newly required genetic markers; EGFR, PTEN, CDKN2A, etc. in WHO2021. RESULTS The cumulative number of cases has reached over 200. Among them, 54 cases were analyzed after WHO2021 revision. The laboratory has maintained the diagnostic platform in which molecular diagnoses are steadily confirmed generally within two weeks. The initial expenditure was exceeded the income obtained from the patient co-payment, however, it has gradually been reduced to running costs alone, then is approaching profitability. After WHO2021 revision, diagnoses could be confirmed by molecular markers obtained from Senshin-Iryo in 38 of 54 cases (70.1%). Among the remaining 16, only 4 (7.4%) cases remained diagnosed as diffuse glioma, NEC, exclusionary after 12 cases in which glioblastoma was confirmed by histopathological diagnosis. CONCLUSIONS Our Senshin-Iryo trial has functioned as a salvage system to overcome the current transition period in which the continuing revision of WHO classification causing a clinical dilemma of healthcare system.

10126-ACT-10 FACTORS AFFECTING THE PATTERN OF RECURRENCE OF IDH-WILDTYPE GLIOBLASTOMA

Abstract INTRODUCTION IDH-wildtype glioblastoma is a quite poor prognosis disease with which almost all cases recur in spite of postoperative radiation and chemotherapy. Survival rates are particularly low after dissemination. METHODS We included 120 patients with IDH-wild type glioblastoma treated at our hospital from January 2015 to December 2019. Patient backgrounds, MGMT promotor methylation status, imaging findings, and recurrence patterns were analyzed. Subependymal, subarachnoid, and spinal dissemination, plus distant metastasis, were defined as non-local recurrence. RESULTS Of 105 patients with a single lesion at diagnosis, 65 had local recurrence and 15 had non-local recurrence. 36 of 65 patients had local recurrence and 12 had non-local recurrence in the second recurrence. 17 of 36 patients had local recurrence and 8 had non-local recurrence in the third recurrence. The cumulative number of non-local recurrences was 54 (45.0%). Surgical ventricular opening and tumor proximity to the ventricles were associated with non-local recurrence (p=0.006, p=0.019). The patients with tumor proximity to the ventricle had non-local recurrence at the first recurrence and those with non-proximity to the ventricle had non-local recurrence at the second or later recurrence (p=0.038). MGMT promotor methylation status and postoperative ischemia were not significantly different from non-local recurrence (p=0.122, p=0.088), but MGMT promotor unmethylation was a risk factor for early non-local recurrence, especially within 1 year (p=0.033). Frontal lobe lesions were less frequently disseminated and more frequently MGMT promotor methylated (p=0.032, p=0.024). Parietal lobe lesions had more MGMT promotor methylation (p=0.006), and temporal lobe lesions had more dissemination (p=0.023). Distant metastasis commonly occurred from the frontal lobe to the ipsilateral or contralateral, and the frontal lobe was frequently associated. CONCLUSION IDH-wildtype glioblastoma is characterized by repeated local recurrence and cumulative non-local recurrence. The pattern of recurrence differs depending on the tumor localization, especially adjacent to ventricle, and MGMT promotor methylation status.

Association of MGMT Promoter and Enhancer Methylation with Genetic Variants, Clinical Parameters, and Demographic Characteristics in Glioblastoma.

The response of glioblastoma (GBM) patients to the alkylating agent temozolomide (TMZ) vitally depends on the expression level of the repair protein O6-methylguanine-DNA methyltransferase (MGMT). Since MGMT is strongly regulated by promoter methylation, the methylation status of the MGMT promoter has emerged as a prognostic and predictive biomarker for GBM patients. By determining the methylation levels of the four enhancers located within or close to the MGMT gene, we recently found that enhancer methylation contributes to MGMT regulation. In this study, we investigated if methylation of the four enhancers is associated with SNP rs16906252, TERT promoter mutations C228T and C250T, TERT SNP rs2853669, proliferation index Ki-67, overall survival (OS), age, and sex of the patients. In general, associations with genetic variants, clinical parameters, and demographic characteristics were caused by a complex interplay of multiple CpGs in the MGMT promoter and of multiple CpGs in enhancer regions. The observed associations for intragenic enhancer 4, located in intron 2 of MGMT, differed from associations observed for the three intergenic enhancers. Some findings were restricted to subgroups of samples with either methylated or unmethylated MGMT promoters, underpinning the relevance of the MGMT promoter status in GBMs.

Magnetic Resonance–Guided Laser Interstitial Thermal Therapy for Recurrent Glioblastoma and Radiation Necrosis: A Single-Surgeon Case Series

To evaluate long-term clinical outcomes among patients treated with laser interstitial thermal therapy (LITT) for predicted recurrent glioblastoma (rGBM). Patients with rGBM treated by LITT by a single surgeon (2013-2020) were evaluated for progression-free survival (PFS), overall survival (OS), and OS after LITT. Forty-nine patients (33 men, 16 women; mean [SD] age at diagnosis, 58.7 [12.5] years) were evaluated. Among patients with genetic data, 6 of 34 (18%) had IDH-1 R132 mutations, and 7 of 21 (33%) had MGMT methylation. Patients underwent LITT at a mean (SD) of 23.8 (23.8) months after original diagnosis. Twenty of 49 (40%) had previously undergone stereotactic radiosurgery, 37 (75%) had undergone intensity-modulated radiation therapy, and 49 (100%) had undergone chemotherapy. Patients had undergone a mean of 1.2 (0.7) previous resections before LITT. Mean preoperative enhancing and T2 FLAIR volumes were 13.1 (12.8) cm3 and 35.0 (32.8) cm3, respectively. Intraoperative biopsies confirmed rGBM in 31 patients (63%) and radiation necrosis in 18 patients (37%). Six perioperative complications occurred: 3 (6%) cases of worsening aphasia, 1 (2%) seizure, 1 (2%) epidural hematoma, and 1 (2%) intraparenchymal hemorrhage. For the rGBM group, median PFS was 2.0 (IQR, 4.0) months, median OS was 20.0 (IQR, 29.5) months, and median OS after LITT was 6.0 (IQR, 10.5) months. For the radiation necrosis group, median PFS was 4.0 (IQR, 4.5) months, median OS was 37.0 (IQR, 58.0) months, and median OS after LITT was 8.0 (IQR, 23.5) months. In a diverse rGBM cohort, LITT was associated with a short duration of posttreatment PFS.

SURG-23. THE INFLUENCE OF PRE-OPERATIVE VARIABLES ON SURVIVAL OF PATIENTS WITH GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is both the most common and most aggressive brain tumor in adults. Despite treatment advances, prognosis remains poor and is influenced by numerous factors. METHODS Patients treated at our institution for glioblastoma were reviewed to identify pre-treatment factors that could increase survival time. 183 treated for high-grade GBM between 2017 and 2021 with complete follow-up data were included. Overall (OS) and progression-free survival (PFS) were calculated with Kaplan-Meier analysis. Cox regression analysis identified the influence of several pre-treatment factors (age, Karnofsky performance status (KPS), radiographic distribution of tumor, and MGMT methylation status) on survival time. RESULTS Average age was 64.9 years. At presentation, 69.9% of patients had KPS > 80. Multifocal tumors occurred in 24.6% of patients, involving an average of 2 distinct regions (either multifocal or unifocal lesions). MGMT methylation was confirmed in 17.5% of patients. Recurrence occurred in 46.4% of patients. Median OS was 21.6 (14.3 – 28.9) months and PFS was 13.4 (10.6 – 16.1) months. MGMT methylation was associated with significantly longer median OS (36.9 vs. 17.0 months, p = 0.012) and PFS (20.0 vs. 10.3 months, p = 0.002) compared to those without. Cox regression confirmed MGMT methylation to be significantly associated with longer PFS (HR -0.883, p = 0.004) and OS (HR -0.785, p = 0.014). Age (HR 0.03, p = 0.003) and KPS > 80 (HR -0.7, p = 0.006) significantly impacted OS, but not PFS. Tumor extent did not impact survival. CONCLUSIONS We found that multifocal tumor, greater number of involved regions. and older age were associated with shorter OS, while MGMT methylation was associated with longer OS. Similar correlations were found for PFS. In upcoming trials, these factors may need to be controlled for between groups in order to accurately determine the effect of new treatments.

NIMG-48. ASSOCIATIONS BETWEEN HETEROGENEITY OBSERVED IN O-6-METHYLGUANINE-DNA METHYLTRANSFERASE (MGMT) ASSAY MEASUREMENTS AND MGMT-BASED IMAGING SIGNATURE BASED ON MRI AND DEEP LEARNING

Abstract BACKGROUND O-6-methylguanine-DNA methyltransferase(MGMT), a gene promoter, corresponds with the efficacy of standard therapy for Glioblastoma (GBM) patients. If a single sample is sequenced, an appropriate diagnosis of MGMT methylation may be hampered by intratumoral heterogeneity. This work proposes a non-invasive in-vivo estimation of MGMT methylation using deep learning and multi-parametric MRI (mpMRI). Further, it quantifies spatial heterogeneity and establishes the associations between MGMT assay measurement and MGMT imaging signature heterogeneity. METHODS For this study, mpMRI structural scans (T1, T2, FLAIR and T1-Gd) of 498 newly diagnosed GBM patients were considered. These patients underwent surgical tumor resection, and MGMT methylation testing was retrospectively collected. By overlaying local and regional 2D patches of size 32 X 32 over the whole tumor on mpMRI scans, a 10-fold cross-validated deep convolutional neural network model was trained considering five layers on the discovery cohort (n=458). The trained model was applied to an independent multi-sample cohort (n=40) to compute the MGMT methylation at the pixel level for imaging-derived maps generation and heterogeneity quantification. The independent cohort included the patients for whom multiple tissue specimens were sequenced for MGMT methylation. RESULTS The patch-based deep model produced an area under the ROC curve of 0.76 (95% CI: 0.71–0.82) for MGMT status prediction. This model showed a strong association between homogeneously MGMT methylated and heterogeneous cases with MGMT Assay measurement of multiple samples with Pearson’s correlation coefficients of 0.64 (p < 0.05) and 0.78 (p < 0.01), respectively. Further, heterogeneity with a significantly higher variance was also observed in heterogeneous cases in developed MGMT imaging signature. CONCLUSION The proposed model efficiently determines MGMT promoter methylation and quantifies GBM spatial heterogeneity non-invasively. Also, it showed a significant association between the heterogeneity observed in MGMT-based imaging signature and MGMT assay measurements.

DNAR-05. EXCEPTIONAL RESPONDERS TO BASE EXCISION REPAIR (BER) INHIBITION FOR RECURRENT GLIOBLASTOMA DISPLAY ENRICHMENT FOR DNA DAMAGE RESPONSE PATHWAYS: RNA SEQUENCING ANALYSIS FROM A MULTICENTER TRIAL

Abstract INTRODUCTION Therapies for glioblastoma recurrence after surgery and chemoradiation are warranted. TRC102 (methoxyamine), a small molecule DNA base-excision repair (BER) inhibitor, reverses temozolomide (TMZ) resistance in preclinical glioma models. We had investigated efficacy of TRC102+TMZ for recurrent glioblastoma (rGBM) through a multicenter trial. This work sought to report translational findings from RNA sequencing performed on patients with available tumor tissue. METHOD: A two-arm, two-stage, non-randomized, Phase II trial of TRC102+TMZ for adults with rGBM named BERT was conducted. Arm 1 included bevacizumab-naive patients at first recurrence, with primary aim of estimating response rates using RANO criteria. If sufficient activity was identified, arm 2 was planned in bevacizumab-refractory patients. Secondary aims were to determine overall survival (OS), progression-free survival (PFS), 6-month PFS, and toxicity. Exploratory aims were correlating clinical outcomes with MPG expression and MGMT methylation. Differential gene expression profiling was analyzed using DESeq2 GSVA (RRID:SCR_015687) and transformed into enrichment scores for 'hallmark of DNA repair' pathways from Molecular Signatures Database (MSigDB, RRID:SCR_016863) to assign mechanistic signatures of therapeutic vulnerability. RESULTS Arm 1 enrolled 19 patients with median of two treatment cycles, but objective responses were not observed. Hence, arm 2 did not open. Median OS was 11.1 months (95%CI 8.2-17.9). Median PFS was 1.9 months (95%CI 1.8-3.7). MGMT promoter was unmethylated in 14/19 patients, and MPG expression was present in 8/14 cases with available tumor tissue. MGMT methylation and MPG non-expression were associated with better OS and PFS. However, two ‘exceptional responders’ had PFS ≥11 months, with MGMT methylated and MPG expressed. RNA sequencing of their tumor tissue demonstrated significant enrichment for DNA damage response (DDR) pathways (MSigDB), chromosomal instability gene signatures (CIN70, CIN25), and proliferative gene signatures (PCNA25). CONCLUSION rGBM patients with elevated levels of MPG and DDR molecular signature may have impaired BER and respond better to TRC102+TMZ.

SURG-03. SURVIVAL STRATIFICATION OF IDH-WILDTYPE GLIOBLASTOMA IN THE MOLECULAR ERA: A RECURSIVE PARTITIONING ANALYSIS INCORPORATING EXTENT OF RESECTION OF CONTRAST-ENHANCING AND NON-ENHANCING TUMORS

Abstract METHODS This multicenter cohort study included a development cohort of 622 IDH-wildtype glioblastoma patients from a single institution (diagnosed between 2001 and 2021) and 445 patients as validation cohorts from two institutions. All patients completed radiotherapy plus concurrent temozolomide and had complete information on IDH mutation and MGMT promoter methylation status. EOR of CE and NE tumors were evaluated. A RPA analysis was performed on behalf of results of the Cox proportional hazards model. The RPA model was compared with a previous RTOG RPA model. RESULTS In the developmental cohort, older age (hazard ratio [HR] = 1.02, P < 0.001), MGMT methylation (HR = 0.51, P < 0.001), KPS (HR = 0.98, P < 0.001), and EOR of CE and NE tumors (GTR of CE tumor and non-GTR of NE tumor, HR = 1.82, P < 0.001; non-GTR of both CE and NE tumor, HR = 2.05, P < 0.001; reference group as GTR of both CE and NE tumor) were independent prognostic factors on multivariable analysis. The novel RPA consisting of age, MGMT methylation, KPS, and EOR of CE and NE tumors revealed greater separation of prognostic classes compared with the previous RTOG RPA model. The prognostic significance of the novel RPA model was subsequently confirmed in the validation cohorts. CONCLUSION The RPA model in IDH-wildtype glioblastoma patients incorporating EOR of CE and NE tumors is a valid model. This simple RPA model has the potential to contribute to improving the accurate assessment of prognostic groups in IDH-wildtype glioblastoma patients and to influence clinical decision-making.

EPCO-28. MULTIPLATFORM MOLECULAR AND FUNCTIONAL GENOMIC ANALYSES OF NF1 MUTANT GLIOBLASTOMA IDENTIFY DISTINCT CO-MUTATION PATTERNS AND DRUGGABLE DEPENDENCIES UNDERLYING SELUMETINIB RESPONSE

Abstract The tumor suppressor NF1 is recurrently mutated in sporadic IDH-wildtype glioblastoma and activates Ras/Raf/MEK signaling, motivating the use of targeted MEK inhibition. Here, we combine multiplatform molecular analysis of NF1 mutant glioblastoma human resection specimens with functional genomic CRISPRi screens to the MEK inhibitor selumetinib. METHODS: We identified 123 consecutive patients treated at the University of California San Francisco between 2016-2022 with NF1 mutant, IDH-wildtype glioblastoma. DNA methylation data was processed using the minfi package, and hierarchical clustering, principal component analysis (PCA), and survival analysis was performed in R. Human NF1 mutant GBM43 and mouse NRAS mutant SB28 GBM cells expressing CRISPRi machinery were used for genome wide selumetinib-resistance screens. Immunoblotting was performed to validate CRISPRi screen hits and selumetinib responses in cell lines. RESULTS: DNA sequencing of human glioblastomas identified 12 recurrent alterations in greater than 10% of cases including cell cycle regulators (CDKN2A/B, TP53, RB1), PI3K signaling (PTEN, PIK3R1, PIK3CA), epigenetic regulators (ATRX, SETD2), and Ras signaling (EGFR, PDGFRA, PTPN11). Methylation data (n=55 tumors) identified three groups marked by differences in MGMT methylation (p=0.04, chi-square test) and CDKN2A/B loss (p=0.01, chi-square test). CRISPRi screens in GBM43 and SB28 cells identified driver phenotypes in cell cycle regulators (TP53, CDKN2A, CDK4), PI3K signaling (PTEN, MTOR), and Ras signaling (RASA2, PTPN11) consistent with co-occurring mutations in the human NF1 mutant glioblastoma cohort. In contrast, mediators of selumetinib response converged on regulators of Ras/Raf/MEK signaling including the Ras effector SHOC2. Biochemical analysis validated SHOC2 repression is sufficient for selumetinib sensitization. CONCLUSIONS: Using a combination of human tumor genomic analysis, functional genetic screens, and biochemical validation, we identify three epigenetic groups of NF1 mutant glioblastoma with distinct co-mutation patterns and validate compensatory Ras/Raf/MEK activation as a conserved mechanism of selumetinib resistance, suggesting combination molecular therapy as a rational therapeutic combination.

INNV-05. OUTCOMES OF RECURRENT GLIOBLASTOMA PATIENTS WITH RADIATION NECROSIS FOLLOWING GAMMATILE BRACHYTHERAPY IMPLANTATION

Abstract Implantation of collagen tile brachytherapy GammaTile (GT Medical Technologies Tempe, AZ) allows for targeting of radiation to residual malignancy following surgical resection of gross disease. Radiation necrosis (RN) can develop after radiotherapy or brachytherapy. We sought to determine outcomes in recurrent glioblastoma patients with RN after treatment with Gammatile. All patients were previously treated with surgical resection followed Stupp protocol chemoradiation. At recurrence, patients underwent repeat surgical resection and implantation of GammaTile. Subsequent MRIs were assessed for stability, presumed RN or progression. Symptomatic RN patients were treated with corticosteroids or bevacizumab. From December 2020 to February 2023, 19 patients were treated. Median time from initial radiation to GammaTile therapy was 270 days. A total of 8 patients (42%) developed RN at a median time of 81 days from GammaTile. In RN patients vs those that did not develop RN, pathological tumor viability was lower (20% vs 50%, p=0.3), age was lower (54 vs 63, p=0.06), tumor volume was lower (10.8 cc vs 51.9 cc, p=0.02), time from initial surgery was longer (470 days vs 299 days, p=0.46), number of GammaTiles placed was lower (5 vs 8, p=0.045), rate of gross total resection (GTR) was higher (100% vs 64%, p=0.04), and MGMT methylation rate was higher (63% vs 11%, p=0.03). No patients in the RN group experienced radiographic progression (0% vs 45%, p=0.009) and 3 (38%) required bevacizumab therapy. Median overall survival from GammaTile surgery in the RN group was 325 days vs 188 days (p=0.03). Our series demonstrates a significant survival benefit in patients who develop RN following GammaTile implantation after glioblastoma recurrence. RN developed in younger patients with smaller tumor volumes, lower pathological tumor viability, and higher rates of GTR and MGMT methylation status.

Association of MRI Volume Parameters in Predicting Patient Outcome at Time of Initial Diagnosis of Glioblastoma.

Patients with glioblastoma (GBM) may demonstrate varying patterns of infiltration and relapse. Improving the ability to predict these patterns may influence the management strategies at the time of initial diagnosis. This study aims to examine the impact of the ratio (T2/T1) of the non-enhancing volume in T2-weighted images (T2) to the enhancing volume in MRI T1-weighted gadolinium-enhanced images (T1gad) on patient outcome. A retrospective audit was performed from established prospective databases in patients managed consecutively with radiation therapy (RT) for GBM between 2016 and 2019. Patient, tumour and treatment-related factors were assessed in relation to outcome. Volumetric data from the initial diagnostic MRI were obtained via the manual segmentation of the T1gd and T2 abnormalities. A T2/T1 ratio was calculated from these volumes. The initial relapse site was assessed on MRI in relation to the site of the original T1gad volume and surgical cavity. The major endpoints were median relapse-free survival (RFS) from the date of diagnosis and site of initial relapse (defined as either local at the initial surgical site or any distance more than 20 mm from initial T1gad abnormality). The analysis was performed for association between known prognostic factors as well as the radiological factors using log-rank tests for subgroup comparisons, with correction for multiple comparisons. One hundred and seventy-seven patients with GBM were managed consecutively with RT between 2016 and 2019 and were eligible for the analysis. The median age was 62 years. Seventy-four percent were managed under a 60Gy (Stupp) protocol, whilst 26% were on a 40Gy (Elderly) protocol. Major neuroanatomical subsites were Lateral Temporal (18%), Anterior Temporal (13%) and Medial Frontal (10%). Median volumes on T1gd and T2 were 20 cm3 (q1-3:8-43) and 37 cm3 (q1-3: 17-70), respectively. The median T2/T1 ratio was 2.1. For the whole cohort, the median OS was 16.0 months (95%CI:14.1-18.0). One hundred and forty-eight patients have relapsed with a median RFS of 11.4 months (95%CI:10.4-12.5). A component of distant relapse was evident in 43.9% of relapses, with 23.6% isolated relapse. Better ECOG performance Status (p = 0.007), greater extent of resection (p = 0.020), MGMT methylation (p < 0.001) and RT60Gy Dose (p = 0.050) were associated with improved RFS. Although the continuous variable of initial T1gd volume (p = 0.39) and T2 volume (p = 0.23) were not associated with RFS, the lowest T2/T1 quartile (reflecting a relatively lower T2 volume compared to T1gd volume) was significantly associated with improved RFS (p = 0.016) compared with the highest quartile. The lowest T2/T1 ratio quartile was also associated with a lower risk of distant relapse (p = 0.031). In patients diagnosed with GBM, the volumetric parameters of the diagnostic MRI with a ratio of T2 and T1gad abnormality may assist in the prediction of relapse-free survival and patterns of relapse. A further understanding of these relationships has the potential to impact the design of future radiation therapy target volume delineation protocols.

BIOM-34. NON-NEOPLASTIC PLASMA EV SUBPOPULATIONS CORRELATE WITH TUMOR SIZE AND OVERALL SURVIVAL IN GLIOBLASTOMA INDEPENDENT OF STEROID USAGE

Abstract 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&amp;lt; 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 (&amp;lt; 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.

EPID-11. THE IMPACT OF THE COVID-19 PANDEMIC ON TREATMENT PATTERNS IN GLIOBLASTOMA

Abstract BACKGROUND The COVID-19 pandemic and subsequent shutdowns led to significant disruptions in medical care and access to treatment for all medical conditions in the US in 2020. Institutional cohorts and survey-based studies have shown that there was significant care disruption for individuals diagnosed with primary brain tumors. In this analysis, we utilized a database containing ~80% of brain tumor diagnoses to assess the national impact of the pandemic on treatment patterns for glioblastoma. METHODS Data on newly diagnosed glioblastoma were obtained from the National Cancer Database, which includes hospital registry data from Commission on Cancer accredited facilities, for diagnosis years 2018-2020. Logistic regression was used to generate odds ratios (OR) of receiving resection, chemotherapy, or radiation after diagnosis in 2020 as compared to 2018-2019, after adjustment for potential confounders, including age at diagnosis, sex, race and ethnicity, insurance status, income, patient distance from diagnosing facility, Charlson comorbidity score, facility type and location, and MGMT methylation. RESULTS There were 7,184 glioblastoma, of which 5,873 were IDH-wt, diagnosed in 2020, a 3% decrease from the 2018-2019 average. For individuals with any newly diagnosed glioblastoma, there was a decrease in odds of resection (OR=0.91, p=0.047). Among those with any glioblastoma and a subset with IDH-wt glioblastoma there was a reduction in odds of radiation (OR=0.87, p=0.004 and 0.85, p&amp;lt; 0.001, respectively) and chemotherapy (OR=0.87, p=0.002 and 0.89, p=0.026, respectively). No decrease in odds of treatment was associated with a prior positive SARS-CoV-2 PCR test. CONCLUSIONS The COVID-19 pandemic caused significant disruption to health care for cancer patients in the US in 2020. For individuals with glioblastoma, in-person treatments decreased in frequency for individuals diagnosed in 2020 as compared to prior years. Further research is required to determine the impact this treatment disruption had on outcomes.

PATH-24. A CASE SERIES OF LONGER THAN EXPECTED SURVIVAL IN IDH-WILDTYPE, MGMT-UNMETHYLATED GLIOBLASTOMA PATIENTS

Abstract Despite interventions with surgery, radiation, and chemotherapy, Glioblastoma (GBM) remains a difficult cancer to treat. Only 6.9% of patients survive five years post-diagnosis1 and the median overall survival is reported to be in the range of 14-15 months.2Glioblastoma patients who harbor an IDH-mutation have a known improved survival rate over their IDH-wildtype counterparts and under the recent WHO 2021 Classification of CNS tumors, IDH-mutational status is now an established criteria for the classification of high-grade gliomas. MGMT methylation status is also a well-described prognostic factor in GBM. MGMT methylated patients have a more favorable prognosis and better response to conventional treatment with temozolomide.3 It is rare but there are some IDH-wildtype, MGMT-unmethylated GBM patients who survive longer than 4 years. Therefore, it is important to identify other potential favorable factors for longer survival in the unfavorable cohort of patients with IDH-wildtype and MGMT-unmethylated GBM. Case Series: Eighty-five GBM patients who had next generation sequencing completed at the University of California, Irvine were reviewed. Of 85 patients, 4 patients (4.7%) were IDH-wildtype, MGMT-unmethylated and lived longer than 4 years. We evaluated the pathology, clinical characteristics, treatment course, and molecular findings of these 4 long term GBM survivors with IDH-wildtype and MGMT-unmethylated status. Discussion: This case series of 4 cases of patients with IDH-wildtype, MGMT-unmethylated GBM with longer than expected survival did not reveal a ubiquitously prevalent molecular marker for long term survival based on review of next-generation sequencing results. 2 out of the 4 patients harbored an uncommon MTAP copy number loss mutation, which may warrant further investigation into the significance of this mutation on prognosis. 3 out of 4 patients received a vaccine-based intervention through clinical trial which may support ongoing investigation into the additional benefit this specific population of GBM patients may have from vaccine-based therapy.

IMMU-39. EFFICACY OF IMMUNE CHECKPOINT INHIBITORS IN GLIOBLASTOMA FROM REAL-WORLD DATA ANALYSIS

Abstract Immune checkpoint inhibitors (ICIs) failed to show efficacy in a randomized trial in glioblastoma (GBM). We utilized the patient-led XCELSIOR platform (NCT03793088) to evaluate ICIs in real-world GBM treatment. XCELSIOR centralizes EMR data from all care sites, creating longitudinal, source-verified data. Identity verification permits accurate overall survival (OS) calculations. As of 6/8/2023, 726 GBM patients were enrolled in XCELSIOR. Median diagnosis date was 1/13/2021 and median age was 56. Seventy-five patients (10%) received ICIs: 53 received pembrolizumab (71%) and 26 received nivolumab (35%) including 12 with ipilimumab (16%). Median days from diagnosis to ICI initiation was 263 days with 22 patients treated at 1st line, 39 at 2nd line, and 14 at 3rd line or later. Data was annotated for 71 patients treated with ICIs and 394 patients not treated with ICIs (no-ICI). Median OS (mOS) from diagnosis for all ICI patients vs. no-ICI was 4.95 years (95% CI 2.18-NR) vs. 1.75 years (1.56-2.03, p &amp;lt; 0.0001). Key features in the ICI cohort were 46% methylated MGMT, 47% gross total resection, 61% exposed to non-SOC agents besides ICI. Creating a propensity score matched cohort for these features, ICI vs. no-ICI had a numerically increased but non-significant mOS of 2.58 years (1.78-NR, n=62) vs. 2.05 years (1.76-2.61, n=113) but a 5-year survival rate of 44% for ICI vs. 16% for no-ICI. Optune use was similar, 39% ICI and 29% no-ICI. Common non-SOC agents used in the ICI cohort were everolimus (11%), osimertinib (8%), lenvatinib (7%), abemaciclib (7%), and neoantigen peptide vaccines (7%). Among unmethylated MGMT patients, mOS was 1.78 years for ICI vs. 1.8 years for no-ICI. These data suggest activity of ICIs for a subset of GBM patients, but do not support use in patients with unmethylated MGMT. Next steps will focus on discrete combination regimens in this evolving data.

DNAR-10. HETEROGENEOUS MGMT PROMOTER METHYLATION IN A MULTIFOCAL GLIOBLASTOMA: A CASE REPORT

Abstract MGMT promoter methylation status is an important predictive biomarker for treatment response in glioblastoma. MGMT unmethylated glioblastoma has elevated levels of MGMT which ultimately leads to chemoresistance. Clinicians consider the MGMT methylation status when treatment recommendations are given, and clinical trials use this marker for the selection of a homogenous group of glioblastoma patients based on prognosis. Here, we present a case of multifocal glioblastoma with heterogeneous MGMT methylation status. A 56-year-old male presented with left leg weakness for 2 months and left arm weakness for 1 month. Imaging revealed a calcified, rim enhancing right temporoparietal cystic lesion and a heterogeneously enhancing right parietal mass. Perilesional edema surrounded and connected the two lesions. The patient underwent gross total resection of the parietal mass and pathology revealed a WHO grade 4 glioblastoma, IDH wildtype, unmethylated tumor. Postoperatively, the patient was neurologically intact. He completed a 6-week long course of concomitant chemoradiation without any significant side effects. Two weeks later the patient developed rapidly worsening headaches, nausea, vomiting, dizziness, and left side weakness. Imaging revealed significant enlargement of the previously unresected temporoparietal cystic lesion with marked increase in surrounding edema and a 2-3 mm right to left midline shift. Patient underwent craniotomy with cyst drainage and tumor resection of the right cystic temporoparietal lesion, demonstrating a WHO grade 4 glioblastoma, IDH wildtype, methylated tumor. Post-operatively the patient was again neurologically intact and started adjuvant temozolomide. MGMT methylation status of glioblastoma can alter recommendations for chemotherapy with temozolomide. MGMT methylation status might vary in distinct parts of the tumor as molecular heterogeneity is a hallmark of glioblastoma. This heterogeneity should be considered by clinicians when treatments are recommended and by clinical trials for glioblastoma patients, especially when multifocal lesions have a distinct radiographic appearance.

CTNI-35. A RANDOMIZED, CONTROLLED TRIAL OF BERUBICIN VS. LOMUSTINE AFTER FIRST-LINE THERAPY FOR GLIOBLASTOMA MULTIFORME (GBM): INTERIM RESULTS OF A PRE-PLANNED ANALYSIS OF OVERALL SURVIVAL (OS)

Abstract Berubicin is an analog of doxorubicin (Dox) that appears to cross the BBB with significant central nervous system (CNS) uptake. RESULTS: of a Phase 1 dose-escalation study with 25 patients evaluable for efficacy showed 1 complete response (14+ years), 1 partial response durable for 12 weeks, and 9 patients with stable disease for a clinical benefit rate of 44%. Our current, potentially pivotal trial for patients with recurrent GBM after first-line therapy is enrolling in the US and Europe. It is unique in that all patients must be Grade 4 IDH WT, classifying these tumors as a poorer prognostic population than Grade 4 IDH mutated. Patients are randomized in a 2:1 design of Berubicin:Lomustine and stratified by MGMT methylation status. The primary endpoint is OS. Data has been presented showing that Berubicin and Lomustine arms are balanced with respect to MGMT status and demographics, with relatively comparable safety profiles (SNO Annual Meeting 2022 Abstract #383142). A pre-planned non-binding futlity analysis of the primary endpoint, OS, is being evaluated when the trial reaches ~30% of expected events (44 events). The interim analysis will be performed by an independent statistician under the supervision of the Data Safety Monitoring Board (DSMB). The futility analysis will be based on the conditional power that the trial will be positive (e.g., statistically significant difference between treatment groups’ OS). If the conditional power is &amp;lt;10% then the DSMB may recommend to end the study; otherwise the study will continue as it is currently planned. The review will also include safety and secondary efficacy endpoint summaries. RESULTS: of this interim analysis will be available for the SNO annual meeting and an evaluation of its outcome will be performed, with conclusions and recommendations for the further development of Berubicin as a therapeutic option for patients with recurrent GBM.