Citations

Abstract Introduction Postoperative radiotherapy is an adjuvant therapy for brain metastases(BrMs) that improves local recurrence(LR). The radiosensitivity index(RSI) is a 10-gene index of pan-cancer molecular radiosensitivity previously validated for predicting radiotherapy response in clinical datasets. In this external validation study, we evaluate RSI as a predictor of local recurrence(LR) after radiotherapy in resected BrM. Methods Patients from University Health Network Toronto(UHN) and McGill University who had a resected BrM and subsequently underwent whole brain radiation(WBRT) or stereotactic radiosurgery(SRS). RSI was calculated using a ranked linear regression model from the weighted expression of 10 radiosensitivity genes, with higher values corresponding to radioresistance. Radiophenotype was determined using a previously validated RSI cutoff of < 0.375 to identify radiosensitive tumors. LR was defined as recurrence in the resection cavity and overall survival(OS) was measured from time of BrM diagnosis. Results 106 BrM patients were included, comprising 29 breast, 37 GI, 33 lung, and 7 melanoma primary sources. Mean age at surgery was 59.2 years, 31.1%(n = 33) were male, 67.9%(n = 72) had WBRT, 23.6%(n = 25) had a subtotal resection, and 30.2%(n = 32) had postoperative chemotherapy. Overall, 34.0%(n = 36) patients had LR. Patients with breast BrMs had median RSI 0.70(IQR:0.55-0.75, 10.3% radiosensitive) with 48.3% LR(n = 14); gastrointestinal BrMs had median RSI 0.43(IQR: 0.35-0.46, 29.7% radiosensitive) and 24.3% LR(n = 9); lung BrMs had median RSI of 0.67(IQR:0.66-0.71, 9% radiosensitive) and 33.3% LR(n = 11); melanoma BrMs had median RSI of 0.72(IQR:0.53-0.92, 0% radiosensitive) and 28.6% LR(n = 2). After adjusting for demographics, primary organ, radiotherapy, resection extent, among others, RSI did not predict LR(p = 0.56) or OS(p = 0.61). Discussion RSI did not predict either LR or OS in patients with surgically resected BrMs status post radiotherapy, further establishing the need for improved predictive scores in this population.

4157 Background: IDH1/2 mutations occur in ~25% of cholangiocarcinomas (CCAs). Preclinical studies suggest synergy between PARP inhibition and PD-L1 blockade. We previously conducted a phase II trial of olaparib plus durvalumab in advanced IDH -mutant CCA. We report matched baseline tissue and serial cell-free DNA (cfDNA)-based correlative analysis of pts samples. The primary objective was to develop a cfDNA methylome signature as a non-invasive biomarker. Methods: This single-arm, open-label phase II study (NCT03991832) enrolled patients with unresectable/metastatic IDH -mutant CCA and ≤2 prior lines of systemic therapy. Patients received olaparib 300 mg orally twice daily and durvalumab 1500 mg IV every 4 weeks. Blood samples were collected at baseline and monthly while on treatment for planned correlatives to examine R2HG, S2HG, and artemin levels using HPLC (High-Performance Liquid Chromatography). Cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) was performed on pts cfDNA samples and case-matched IDH -wildtype pts. Formalin-fixed, paraffin-embedded blocks of baseline tumor biopsies were used for multispectral fluorescent IHC (FL-IHC) using a 6-marker panel composed of pan-cytokeratin, CD4, CD8, CD163, PD-L1, and CD68. Results: Ten patients were enrolled (median age 63.5 y; 50% female; 90% received prior platinum). Median treatment duration was 1.95 mo (range 1.8–13.5). No complete or partial responses were observed. DCR was 30% (3 patients with stable disease); one patient remained on therapy for 13.5 mo before progression. Median PFS was 1.97 mo (95% CI 1.73–3.93). HPLC from baseline plasma showed lower baseline artemin levels correlated with stable disease (p=0.038), while R2HG/S2HG ratios showed no association with outcome. FL-IHC showed enriched immune cell populations, specifically CD4 + T cells (p=0.0032) and a trend for CD68 + macrophages (p=0.062), in the tumor stroma compared with tumor core. 54 plasma samples (from 8 patients) were profiled with cfMeDIP-seq along with 8 IDH -wildtype controls. Using top 100 differentially methylated regions, the circulating tumour DNA methylome exhibited a highly specific signature, accurately discriminating IDH -mutant and IDH -wildtype CCA regardless of treatment status. The specific differentially methylated gene pathways between groups were comprehensively examined in both on-treatment and baseline plasma samples. Conclusions: Advanced IDH -mutant CCA harbors an immune-excluded phenotype, which may underlie primary resistance to immunotherapy. Plasma cfDNA methylome signature can accurately discriminate CCA with IDH mutations. Clinical trial information: NCT03991832 .

Abstract Background Meningiomas exhibit clinical heterogeneity. Radiotherapy (RT) remains the only adjuvant therapy, but tumor-control is variable, and biomarkers are limited. NRG/RTOG-0539 is the first prospective phase 2 trial to stratify meningioma patients for adjuvant RT based on clinical risk. Here, we apply modern molecular tools to this cohort and identify correlates of RT response. Methods Tumor tissue from 100 RTOG-0539 patients was profiled using DNA methylation arrays, RNA sequencing, and whole-exome sequencing. Recurrence scores, Molecular Groups, gene expression, and copy number alterations were compared across clinical groups and between RT responders and non-responders; non-response was defined as progression or death within 3 years. Results Modern grading criteria, including brain invasion, TERT mutation, CDKN2A/B deletion, and 1p/1q status, would reclassify 10% of tumors and alter treatment group assignment in 7%. Non-responders to RT exhibited more frequent 1p and 14q loss, and more copy number alterations. Transcriptomic and epigenetic profiling revealed immune-related signatures in responders and cell cycle–related pathways in non-responders, several of which overlapped with targets of vorinostat, a histone deacetylase inhibitor previously validated in aggressive meningioma models. The Proliferative Molecular Group was an independent predictor of post-RT recurrence in multivariable analysis, outperforming WHO grade. Conclusion Multi-omic analysis of the NRG/RTOG-0539 cohort shows that updated WHO grading criteria, incorporating molecular and cytogenetic features, improve risk stratification. However, molecular classification, particularly the Proliferative group, remains an independent and stronger predictor of RT response. These findings support integrating molecular biomarkers alongside modern grading frameworks to guide treatment and trial design in meningioma. Clinical Trial Information NCT00895622.

Abstract Purpose: Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor, with a median survival of only 15 months. Effective therapeutic advances remain limited, with few clinical trial patients exhibiting robust responses to experimental therapies. This has led to efforts to identify molecular biomarker signatures capable of stratifying patient responses either prognostically (pre-treatment) or diagnostically (post-treatment). This study aims to assess the robustness and reproducibility of published transcriptional GBM biomarker signatures for predicting clinical outcomes in GBM trials. Methods: We analyzed published RNA-seq data from 4 clinical trials involving oncolytic virotherapy and immune checkpoint inhibitors, as well as multiple cohorts of patients treated via the Stupp protocol. The performance of reported immune gene signatures was evaluated across all datasets using both grouped and continuous survival modeling. Intra-patient signature variation between different biopsy sites and timepoints was assessed using longitudinal, multi-site sampling collected by the Break Through Cancer Accelerating GBM Therapies TeamLab. LASSO, multivariate Cox regression, and risk scoring with FDR adjustment were used to identify novel signatures with cross-cohort utility. Key Findings: No single published signature consistently predicted survival across all analyzed trials. A post-treatment ssGSEA antitumor cytokine signature that was associated with survival in the CAN-3110 OV trial (R = 0.74, p < 0.01) was also significantly associated with improved survival in an adjuvant anti-PD1 trial (R = 0.75, p = 0.03) and neoadjuvant anti-PD1 trial (R = 0.30, p = 0.05), but not in standard-of-care controls. PAM clustering of MCP immune signatures, as reported for the DNX-2401 OV+Anti-PD1 trial, also stratified post-treatment survival in the CAN-3110 OV trial (p < 0.01), with the coldest TME subtype consistently predicting worse survival. However, the prognostic value of the most immune enriched TME subtype was inconsistent and did not extend to the Anti-PD1 trials. Furthermore, multi-site, longitudinal biopsy samples revealed marked heterogeneity in biomarker signatures between biopsies from individual patients, even when assessed at the same timepoint. We’ve identified several candidate transcriptomic signatures that show promise for generalizable prognostic value in GBM patient cohorts and are characterizing their spatiotemporal variability. Conclusions: While no individual immune signature is universally predictive in GBM immunotherapy, certain post-treatment cytokine signatures and TME stratifications are significant in multiple immunotherapy contexts, though not in standard-of-care patient cohorts. Marked spatiotemporal heterogeneity in these signatures underscores the need for composite prognostic markers resilient to sampling variance. Citation Format: Christopher M. Jannotta, C Zoe Linke, Charles A. Whittaker, Vikas Patil, Accelerating GBM Therapies TeamLab, Farshad Nassiri, Gelareh Zadeh, E Antonio Chiocca, Alexander L. Ling. Assessing biomarker reproducibility for glioblastoma patient response stratification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1055.

Abstract Background DNA methylation profiling can be used to robustly predict postsurgical outcomes and response to radiotherapy (RT) for meningioma patients. To allow for seamless integration of these complementary models into clinical practice, a practical framework is needed. Methods We leveraged a cohort of nearly 2000 surgically-treated meningiomas with DNA methylation profiling and clinical outcomes data. Existing methylation-based prediction models were dichotomized to yield four risk groups: low and high recurrent risk, each with RT sensitive and resistant subgroups. Risk groups were correlated with progression-free survival in the context of existing biomarkers including extent of resection and WHO grade. Results We first demonstrated that all risk groups benefit from gross total resection. All “high-risk, RT sensitive” tumors (n = 306, 15.7%) also benefited from adjuvant RT: after GTR, median PFS increased from 4.68 (4.13-9.48) years to not reached (P = .003); after subtotal resection (STR), from 2.12 (1.59-3.02) to 4.09 (3.41-not reached) years (P = .004). “Low-risk, RT sensitive cases” (n = 1207, 61.8%) also benefited from RT after STR (median PFS 7.39 (6.66-12.8) vs. 16.53 (10.35-not reached) years, P = .03), suggesting that RT be considered in these patients. Neither “low-risk RT resistant” (n = 84, 4.3%) nor “high-risk RT resistant” (n = 356, 18.2%) cases benefited from RT, and the latter group was associated with universally poor outcomes. Conclusions We identify methylation-defined risk groups of meningioma for which additional benefit is gained from adjuvant RT, leading to a clinical decision-making framework for straightforward integration of molecular models into clinical practice.

Abstract A new mouse model for the classical subtype of human glioblastoma (GBM) has been bred using Cre-mediated EGFRvIII overexpression and homozygous p19-ARF deletion, the mouse homolog of human p14ARF/CDKN2A, in GFAP expressing cells. Transgenic mice develop intraparenchymal and/or leptomeningeal brain lesions with some spinal cord invasion as early as 1 month old and 95% of mice die by 6 months due to hydrocephalus and/or paralysis. Mice with high grade tumors have worse survival and similar features to human classical GBM such as necrosis, high levels of mitosis and infiltration of tumor cells into normal brain. Immunohistochemical analysis confirms EGFRvIII overexpression and p19-ARF loss in Cre-expressing cells, along with patchy positive GFAP and high proliferation by Ki67. Bulk RNA sequencing reveals GEC3 tumors cluster closely with human classical GBM and have significant upregulation of downstream markers in the JAK/STAT and P13K/AKT pathways. Adherent and neurosphere primary culture of dissociated tumors indicate that tumor cells maintain EGFRvIII expression in culture and generate xenograft tumors by 3 weeks after intracranial injections into NODSCID mice. Xenograft tumors are reminiscent of the primary tumor, with similar histopathological features and immunohistochemical staining. Our spontaneous glioma model is a powerful tool for future studies focused on the role of the immune microenvironment on glioma recurrence and resistance.