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Background: Meniscal preservation has been demonstrated to contribute to long-term knee health and has been a successful intervention in isolation and in patients with anterior cruciate ligament reconstruction (ACLR). The long-term results of meniscal repair in the setting of revision ACLR have yet to be documented. Purpose: To report the incidence of meniscal repair failures at the 6-year follow-up in a cohort of patients who underwent concurrent revision ACLR and primary meniscal repair. Study Design: Prospective cohort study; Level of evidence, 2. Methods: All revision ACLRs with concomitant primary meniscal repair cases from a multicenter group between 2006 and 2011 were selected. Six-year follow-up was obtained to determine whether any subsequent surgery had occurred since their initial revision ACLR. If so, operative reports were obtained, whenever possible, to verify pathological condition and treatment. Results: In total, 221 patients from 1234 revision ACLRs underwent concurrent primary meniscal repairs (18% of the cohort). There were 238 repairs performed: 173 medial and 65 lateral. The majority of these repairs (n = 181; 76%) were performed with an all-inside technique. Six-year surgical follow-up was obtained in 77% (171/221) of the cohort, or 189 of 238 (79%) of the repairs (136 medial, 53 lateral). The meniscal repair failure rate, defined as reoperation, was 16% (31/189) at 6 years. Of the 31 failures, 28 were medial (24 all-inside, 4 inside-out; 28/136 = 20.6% failure rate) and 3 were lateral (2 all-inside, 1 inside-out; 3/53 = 5.7% failure rate). Three medial failures were treated in conjunction with a subsequent repeat revision ACLR. Medial tears underwent reoperation for failure at a significantly higher rate than lateral tears (20.6% vs 5.7%; P = .01) and had a significantly shorter survival time compared with lateral tears ( P = .02). No difference was found between the failure and nonfailure groups when it came to tear type, tear length, repair technique utilized, suture/implant type, or number of sutures used between the 2 groups. Conclusion: Meniscal repair in the revision ACLR setting has a 16% failure rate at 6 years. Failure rates for medial tears (20.6%) were found to be higher than that for lateral tears (5.7%), which aligns with previous studies in both the revision and primary ACLR setting.

Chronic traumatic encephalopathy (CTE) is a neurodegenerative disease associated with exposure to repetitive head impacts (RHI). In CTE, hyperphosphorylated tau (p-tau) aggregates are found in neurons at the depth of cortical sulci close to the gray matter/white matter (GM/WM) boundary. To date, CTE can only be diagnosed postmortem by neuropathological examination. Traumatic encephalopathy syndrome (TES) is the clinical syndrome purported to be associated with CTE pathology. The aim of this study is to investigate microstructural properties at the GM/WM boundary in individuals with a history of exposure to RHI and clinical features of CTE (i.e., TES). Diffusion magnetic resonance imaging (dMRI), TES diagnoses, and cerebrospinal fluid (CSF) biomarkers were acquired from 165 male former American football players (age: 57.29 ± 8.23 years) from the DIAGNOSE CTE Research Project, a multicenter, observational cohort study. Fractional anisotropy (FA) was measured at the GM/WM boundary of the whole brain. In addition, a widely used method (tract-based spatial statistics [TBSS]) was applied to measure FA of central WM. We used analyses of covariance to test associations between FA and TES. Furthermore, we used linear regressions to test associations between FA and nine CSF biomarkers (i.e., p-tau-181, -217, -231, total tau, amyloid β [Aβ] 1–40 , Aβ 1–42 , glial fibrillary acidic protein [GFAP], neurofilament light [NfL], and soluble triggering receptor expressed on myeloid cells-2 [sTREM2]). We report an association between higher FA at the GM/WM boundary and higher levels of certainty for CTE pathology ( F (1, 147) = 5.781, 95% confidence interval (CI) = 0.0003–0.003, p = 0.035) as well as neurobehavioral dysregulation ( F (1, 148) = 7.559, 95% CI = 0.001–0.009, p = 0.020), and functional dependence/dementia ( F (1, 148) = 5.046, 95% CI = 0.0004–0.006, p = 0.039). In addition, we report an association between higher FA at the GM/WM boundary and higher CSF p-tau-181 (β = 0.272, 95% CI = 0.078–0.466, p = 0.029) and p-tau-217 (β = 0.295, 95% CI = 0.102–0.488, p = 0.027). FA of the central WM was not associated with TES diagnoses. Taken together, these findings suggest that dMRI at the GM/WM boundary could be used to investigate microstructural alterations suggestive of tau pathology-associated neurodegeneration in individuals with TES, the clinical presentation of CTE. Future studies are needed to validate this approach and to identify clinically useful cutoff values for dMRI metrics.

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults, with a dismal 5-year survival rate of ~6.5%. Vasogenic cerebral edema is a severe complication that occurs in the majority of GBM patients, leading to a significant increase in intracranial pressure, neurological deficits, and increased mortality rates. GBM-associated edema is almost exclusively managed by the corticosteroid dexamethasone, the use of which is associated with immunosuppression and interference with radiation therapy. While there is considerable research on edema formation, little is known about how edema resolution occurs in GBM, particularly in response to dexamethasone or other anti-edema therapies. This gap in understanding highlights the need for new approaches to treat edema effectively. The water channel aquaporin-4 (AQP4), a key player in the brain’s water homeostasis, is upregulated in GBM compared to the normal brain. To investigate the role of AQP4 in GBM-associated edema, we used the RCAS/tv-a system to generate de novo GBM with different driver mutations in Aqp4 knockout (KO) and wild-type (WT) mice. Our results indicated that Aqp4 KO tumor-bearing mice had shortened survival and increased edema content. Additionally, Aqp4 loss decreased astrocyte coverage specifically in the tumor core but not the tumor periphery. Our single cell RNA-seq data suggested that Aqp4 loss led to an astrocytic shift towards a less reactive, progenitor-like state, while also decreasing the myeloid and CD8+ T cell population in the tumor microenvironment. Interestingly, tumor-bearing mice treated with anti-VEGFA antibody, which has anti-edema properties, showed increased expression of Aqp4 and reduced vessel leakage. This suggests that AQP4 plays a critical role in mediating the effects of anti-VEGFA therapy. Overall, this study highlights the important role of AQP4 in the resolution of edema, while also revealing its additional effects on astrocyte activation and the immune microenvironment in GBM.

Abstract Pediatric, adolescent and young adult patients (Ped/AYA; 0–39 years) with meningiomas are underrepresented in neuro-oncology research, despite meningiomas being the second most common primary brain tumor in this age group. While DNA methylation-based models have advanced classification and prognostication in adults, their utility in younger populations remains uncertain. To address this gap, we aimed to evaluate the performance of current DNA methylation-based classification and recurrence prediction models in Ped/AYA patients with meningiomas and identify age-associated molecular differences. As part of this study, we analyzed 1,568 meningioma samples with DNA methylation and clinical data, including subsets with whole-exome and RNA sequencing. Patients were grouped into Ped/AYA (≤39 years, n = 213) and adult (>39 years, n = 1,355) cohorts. We assessed classifier performance, performed subgroup-specific model retraining, and compared molecular profiles across age groups. Our results showed that molecular classification differed significantly between age groups, with Ped/AYA tumors less frequently belonging to the most aggressive Proliferative Molecular Group. The DNA methylation-based recurrence model performed well in adults (AUC = 0.83) but poorly in the Ped/AYA cohort (AUC = 0.57). However, retraining the model on only younger patients improved performance (AUC = 0.75). In addition, Ped/AYA tumors exhibited significantly fewer chromosomal alterations, including lower frequencies of 1p, 6q, and 14q loss (p<0.05). Taken together, these findings indicate that predominantly adult-trained methylation models underperform in younger meningioma patients due to both biological divergence and exclusion from training datasets. Our findings underscore the need for age-specific molecular tools to improve prognostication and support personalized care across the lifespan in neuro-oncology.

Abstract The WHO 2021 classification of central nervous system tumors incorporated molecular markers to better define diffuse glioma subgroups. We analyzed array-based copy number, targeted next-generation sequencing, germline genotyping, and Illumina EPIC array methylation data from 404 primary IDH-mutant astrocytomas to further understand the relationship between copy number and outcomes. Loss of chromosome arm 13q was associated with poor overall survival after adjusting for age, sex, tumor grade, and treatment status (hazard ratio [HR]=2.04 (95% confidence interval: 1.25-3.33), p=0.004). Chromosome 13q loss was also significantly associated with the total number of copy number alterations (p<0.001). Loss of arm 11p was associated with poor survival in the adjusted analysis (HR=2.29 (1.38-3.79), p=0.001). Unsupervised clustering of the raw copy number data identified 12 clusters, where each cluster was defined by distinct copy number alteration(s). Clusters that contained 13q loss exhibited worse overall survival compared to other clusters. The clusters were significantly associated with age at diagnosis (p=0.015), tumor grade (p<0.001) and copy number complexity (p<0.001). The clusters with a high proportion of grade 4 tumors and with the highest complexity had the poorest overall survival. These findings highlight the prognostic relevance of chromosome 13q and 11p losses in IDH-mutant astrocytomas and support the integration of additional copy number alterations into molecular classification frameworks of IDH-mutant astrocytomas to improve risk stratification and patient management.

Abstract Brain metastases (BMs) are a common and often fatal progression of systemic cancers, affecting up to 25% of patients. Despite advances in surgical resection and radiotherapy, the median survival remains limited to 10–16 months. While genomic profiling has enabled the development of targeted therapies, there remains a paucity of prognostic tools for clinical use. To address this, we explored the utility of DNA methylation profiling—an epigenetic marker increasingly recognized for its diagnostic and prognostic value. We profiled fresh-frozen tissue from 327 surgically resected BM samples of lung, breast, melanoma, and gastrointestinal (GI) origin using the Illumina Infinium MethylationEPIC BeadChip array. Unsupervised analysis via partitioning around medoids (PAM) clustering identified five robust groups. Four of these correlated closely with primary tumor origin. However, a distinct “Poly-Origin Cluster” emerged, comprising tumors from multiple primary sites with a shared epigenetic signature. Poly-Origin tumors showed improved survival outcomes compared to origin-aligned clusters (20.2 vs. 10.1 months, p=0.0018), independent of clinical covariates. Notably, these tumors were enriched for immune cell infiltration—including CD14+ macrophages, CD19+ B-cells, and CD56+ NK cells—based on deconvolution analysis, and exhibited reduced genomic instability as measured by copy number variation. These tumors were enriched for pathways related to cell signaling. We validated the prognostic significance of the Poly-Origin methylation signature in a publicly available independent cohort (n=96), and found that high signature concordance correlated with extended survival. Finally, we demonstrated the feasibility of detecting this signature in plasma-derived cell-free DNA using cfMeDIP-seq, with high classification accuracy (AUC = 0.98). These findings reveal a clinically meaningful subtypes of BM not captured by tissue origin or mutation status alone. Our work suggests that DNA methylation profiling may be a powerful tissue- and liquid biopsy–based tool that holds promise as a non-invasive prognostic biomarker in the management of brain metastasis.

Abstract A major barrier in understanding adult diffuse gliomas lies in the inability to track tumor evolution in real time. To address this, we employed a novel method known as continuous lineage tracing, which integrates CRISPR/Cas9-based expressed DNA barcoding with single-cell RNA sequencing, enabling a phylogenetic approach to studying tumor development. Patient-derived glioma-initiating cell lines were engineered with expressed barcodes targeted by CRISPR/Cas9 and implanted into mice to create intracranial xenografts. Tumors underwent single-cell RNA sequencing; expressed barcodes were used to infer clonal relationships, and transcriptomic profiles enabled cell state classification. Phylogenetic lineage trees were reconstructed using lineage inference algorithms to characterize cell fitness, expansion, and plasticity. Our analysis uncovered extensive intra-clonal cell state heterogeneity, indicating active phenotype switching prior to therapy. We identified consistent transcriptional programs associated with tumor engraftment and in vivo clonal advantage. Lineage tracing revealed gene expression signatures linked to key phenotypes: fitness, enriched for neural-mesenchymal and injury-response pathways; expansion, associated with RNA splicing; and plasticity, correlated with cell cycle and DNA repair programs. Glioma stem cells appeared to span a transcriptional continuum from undifferentiated, high-fitness states to more differentiated, low-fitness states, with high-fitness cells potentially representing transitional phenotypes. We then validated these findings in a cohort of 185 surgically resected gliomas with matched bulk RNA and proteomic data. Phylogenetic gene signatures differed markedly between IDH-mutant and IDH-wildtype tumors. When stratified by tumor type, both fitness and expansion signatures were significantly associated with overall survival in GBM (median 12.6 months, p=0.041) and oligodendrogliomas (median 66 months, p=0.027). This study demonstrates the utility of continuous lineage tracing to reconstruct tumor evolution and identify transcriptional programs linked to tumor growth and prognosis. Our approach provides a powerful framework for dissecting glioma biology and identifying potential therapeutic vulnerabilities.

Abstract INTRODUCTION Recurrent IDH-mutant gliomas pose a significant therapeutic challenge, with limited treatment options following progression after standard therapy. Combining PARP inhibitors with immune checkpoint blockade has been proposed as a synergistic strategy in IDH-mutant high-grade gliomas, leveraging vulnerabilities in homologous recombination repair and increased PD-L1 expression following PARP inhibition. OBJECTIVE This phase II trial (NCT03991832) evaluated the combination of the PARP inhibitor olaparib and the PD-L1 inhibitor durvalumab in patients with recurrent IDH-mutant glioma. We also investigated the potential of the plasma tumor methylome as a non-invasive biomarker of treatment response. METHODS Twenty-nine patients (median age 40.5 years; 41% female) were enrolled between January 2020 and February 2023. All patients received olaparib (300 mg twice daily) and durvalumab (1,500 mg IV every four weeks) until radiographic or clinical progression. Plasma samples were collected at baseline and monthly, and cell-free methylated DNA immunoprecipitation sequencing (cfMeDIP-seq) was performed. RESULTS The objective response rate was 10%, and median overall survival was 9.3 months. Longitudinal cfMeDIP-seq profiling enabled development of a circulating methylome classifier that accurately distinguished responders from non-responders. Integration with matched tumor transcriptomic and methylation data revealed enrichment of immune and DNA repair pathways in responders. Whole-exome sequencing identified no consistent mutational correlates. Spatial transcriptomic analysis demonstrated a more interactive, immune-rich tumor microenvironment and reduced malignant cell state diversity in responders. CONCLUSION This study supports the safety of combined PARP and PD-L1 blockade in recurrent IDH-mutant glioma and highlights the plasma methylome as a promising non-invasive biomarker of therapeutic response.

Abstract Meningiomas are the most common primary intracranial tumors and exhibit clinical heterogeneity. Radiotherapy (RT) remains the only adjuvant therapy, but response is variable and biomarkers are limited. 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 uncover correlates of RT response. To do so, 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. Using this approach, 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%. Notably, non-responders to RT had more frequent 1p and 14q loss, and greater copy number burden. In addition, transcriptomic and epigenetic profiling revealed immune-related signatures in RT responders and upregulation of cell cycle–related pathways in RT non-responders, several of which overlapped with targets of vorinostat, a histone deacetylase inhibitor validated in aggressive meningioma models. Of particular importance, the Proliferative Molecular Group was an independent predictor of post-RT recurrence in multivariable analysis, outperforming WHO grade. In conclusion, multi-omic analysis of the 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.

Abstract INTRODUCTION While TERT promoter mutation (TPM) has been established as a marker of clinically aggressive meningiomas, this alteration is rare and found in less than 5% of all cases. However, a larger subset of meningiomas may exhibit aberrant TERT expression in the absence of TPMs. This study investigated the effect of TERT gene expression on clinical outcome in meningioma patients. METHODS Clinical and molecular data were retrospectively collected on 1241 meningiomas, split into a Toronto discovery cohort and a multi-institutional validation cohort. Sanger sequencing and bulk RNA sequencing were used to determine TPM status and TERT gene expression. The effect of TERT expression on progression-free survival (PFS) was assessed using Kaplan-Meier and Cox regression analysis. RESULTS While meningiomas with TPM showed expectedly higher TERT gene expression compared to wildtype (TP-WT) cases (p<0.0001), TERT expression was still detected in 28.7% (157/547) of TP-WT meningiomas. Meningiomas with TERT expression showed significantly worse PFS compared to meningiomas without any TERT expression. In fact, WHO grade 1 meningiomas with TERT expression had PFS outcomes resembling WHO grade 2 meningiomas lacking TERT expression (p=0.59). In turn, WHO grade 2 meningiomas with TERT expression had clinical outcomes similar to WHO grade 3 meningiomas without TERT expression (p=0.42). Furthermore, the proportion of meningiomas expressing TERT as well as overall TERT expression levels increased with increasing WHO grade. Multivariable analysis showed that TERT expression was significantly associated with worse PFS even when controlling for other known predictors of clinical outcome including TPM, CDKN2A/B loss, 1p/22q status and WHO grade (HR 1.85 [95% CI 1.33-2.57], p=0.00024). CONCLUSION TERT expression is a novel independent biomarker of outcome for meningiomas identifiable in up to one-third of cases that may be utilized to reclassify tumours to a higher WHO grade.