Lower-grade glioma (LGG) is a diffuse infiltrative tumor of the central nervous system, which lacks targeted therapy. We investigated the role of Podocan-like 1 (PODNL1) methylation in LGG clinical outcomes using the TCGA-LGG transcriptomics dataset. We identified four PODNL1 CpG sites, cg07425555, cg26969888, cg18547299, and cg24354933, which were associated with unfavorable overall survival (OS) and disease-free survival (DFS) in univariate and multivariate analysis after adjusting for age, gender, tumor-grade, and IDH1-mutation. In multivariate analysis, the OS and DFS hazard ratios ranged from 0.44 to 0.58 (p < 0.001) and 0.62 to 0.72 (p < 0.001), respectively, for the four PODNL1 CpGs. Enrichment analysis of differential gene and protein expression and analysis of 24 infiltrating immune cell types showed significantly increased infiltration in LGGs and its histological subtypes with low-methylation levels of the PODNL1 CpGs. High PODNL1 expression and low-methylation subgroups of the PODNL1 CpG sites were associated with significantly increased PD-L1, PD-1, and CTLA4 expressions. PODNL1 methylation may thus be a potential indicator of immune checkpoint blockade response, and serve as a biomarker for determining prognosis and immune subtypes in LGG.

Background: Tumour mutation burden (TMB) is an emerging pan‐cancer biomarker with predictive value for immune checkpoint inhibitor (ICI) outcomes, yet evidence is inconsistent due to methodological variability and cut‐off thresholds. This systematic review and meta‐analysis evaluated the impact of TMB on overall survival (OS) and progression‐free survival (PFS) across solid tumours in ICI‐treated cohorts and its predictive relevance in non‐ICI‐treated cohorts. Methods: Following PRISMA 2020 guidelines, we searched PubMed, Scopus, ScienceDirect and Cochrane for studies published between 2010 and 2024 reporting hazard ratios (HRs) and 95% confidence intervals (CIs) for OS and PFS in high‐ versus low‐TMB cohorts. High and low TMB were defined by study‐specific cut‐offs, and ultra‐high TMB was defined as the top 20% of cohort‐specific values. Study quality was assessed with the Newcastle‐Ottawa Scale; heterogeneity with I ² ; publication bias with funnel plots/Egger's test; and robustness by leave‐one‐out analysis. Results: 5278 patients across 28 studies were analysed. High TMB, defined by cohort‐specific cut‐offs, was significantly associated with improved OS and PFS, particularly in non‐small cell lung cancer (OS: HR = 0.56), selected gastrointestinal cancers (OS: HR = 0.36), and advanced/recurrent tumours (OS: HR = 0.52). Benefits were greatest in ICI‐treated patients, especially with combined anti‐PD‐L1/PD‐1 and anti‐CTLA‐4 therapy (OS: HR = 0.47; PFS: HR = 0.50). Chemotherapy‐treated cohorts also showed better outcomes, but less consistently (OS: HR = 0.60; PFS: HR = 0.55). Ultra‐high TMB had better OS than the universal 10 mut/Mb cut‐off (HR = 0.44 vs. 0.58). Non‐beneficial associations were observed in glioma and penile squamous cell carcinoma, highlighting disease‐specific variability. Sequencing platforms and cut‐off definitions remained sources of heterogeneity. Conclusion: TMB demonstrates prognostic relevance and predictive utility in a histology‐ and treatment‐context‐dependent manner, with the most consistent associations in selected ICI‐treated tumours. Associations in non‐ICI‐treated cohorts were weaker and inconsistent, indicating putative predictive value. Standardising TMB assessment and refining relevant thresholds are essential for optimising its role in precision oncology. Trial Registration: PROSPERO Registration Number: CRD42024608809

Background Tumour mutation burden (TMB) is an emerging pan-cancer biomarker with potential predictive value for immune checkpoint inhibitor (ICI) therapy outcomes. However, its prognostic significance remains inconsistent due to methodological variability and differing cut-off thresholds. This systematic review and meta-analysis evaluated the impact of TMB on overall survival (OS) and progression-free survival (PFS) across solid tumours. Methods Following PRISMA 2020 guidelines, we systematically searched PubMed, Scopus, ScienceDirect, and Cochrane databases for studies published between 2010 and 2024. Eligible studies reported hazard ratios (HRs) and 95% confidence intervals (CIs) comparing OS and PFS in high versus low TMB cohorts. Heterogeneity was assessed using the I² statistic, and publication bias via funnel plots and Egger’s test. Results A total of 5,278 patients across 28 studies were analysed. High TMB was significantly associated with improved OS and PFS, particularly in non-small cell lung cancer (OS: HR = 0.56), gastrointestinal cancers (OS: HR = 0.36), and advanced/recurrent tumours (OS: HR = 0.52). Survival benefits were most pronounced in ICI-treated patients, especially those receiving combination anti-PD-L1/PD-1 and anti-CTLA4 therapy (OS: HR = 0.47; PFS: HR = 0.50). Ultra-high TMB cases had superior outcomes (OS: HR = 0.44) compared to a universal 10 mut/Mb cut-off (OS: HR = 0.58). Variability in TMB measurement across sequencing platforms highlights the need for standardisation. Conclusion High TMB is a strong prognostic and predictive biomarker in ICI-treated cancers, yet methodological inconsistencies hinder clinical implementation. Standardising TMB assessment and refining clinically relevant thresholds are essential for optimising its role in precision oncology. PROSPERO registration number The protocol of this systematic review is registered on PROSPERO (CRD42024608809).

PIWI proteins, a conserved Argonaute subfamily of proteins that interact with PIWI-interacting RNAs (piRNAs), have been implicated in cancer development. However, their roles in glioblastoma (GBM), particularly in glioblastoma stem cells (GSCs), a therapy-resistant subpopulation of malignant cells that drives tumor progression, remain insufficiently defined. We integrated bulk RNA sequencing, single-nuclei RNA sequencing, and RT-qPCR across large and well-defined GBM cohorts (146 patients) to generate the first comprehensive transcriptomic, tumor-, and cell-state-resolved analysis of PIWIL1–4 expression. PIWIL2 and PIWIL4 were consistently expressed in GBM, whereas PIWIL1 and PIWIL3 were largely absent. Single-nuclei RNA sequencing showed that PIWIL2 was enriched in the mesenchymal-like 2 (MES2) state associated with hypoxia, a condition linked to cancer stemness. Using 24 paired patient-derived GSC and non-GSC cultures, we found that PIWIL2 transcript expression was consistently enriched in GSCs and decreased upon differentiation. Transient siRNA-mediated silencing of PIWIL2 reduced GSC viability and was associated with reduced sphere-forming capacity, decreased total normalized piRNA counts, and altered expression of specific piRNAs. PIWIL4 was expressed in both GSC and non-GSC cultures and was enriched in MES1-, AC-, and NPC2-like states. Together, these findings provide a comprehensive characterization of PIWI family expression in GBM and, through integrated transcriptomic and functional analyses, identify PIWIL2 as the PIWI family member most consistently linked to the GSC phenotype.

Charlie Teo Foundation Brain Tumour Bank (CTFBTB) was started by Charlie Teo Foundation (CTF), an Australian charity funding brain cancer research. CTFBTB was established under the CTF’s research strategy of More Data – to produce and share high-quality, well-annotated, biological and clinical data that has the potential to drive further innovation and scientific breakthroughs. CTFBTB collects samples of human tissue, live cells, DNA and blood to be used for brain cancer research. CTFBTB also has a cell line repository of real-world brain tumours. Researchers can apply to use these valuable and accurate models that enable more refined analysis of the mechanisms that regulate individual patient response to treatment and allow for the further development of models for precision medicine.