Alterations in 5hmC and TET2 expression in bladder cancer and their associations with clinicopathological features and patient outcome

Abstract Bladder cancer (BLCA) remains a major clinical challenge due to its high recurrence rate and the limited availability of reliable prognostic biomarkers. Since epigenetic dysregulation is a hallmark of urothelial carcinogenesis, we investigated the expression patterns, clinicopathological associations, and prognostic significance of 5-hydroxymethylcytosine (5hmC) and TET2, a key regulator of active DNA demethylation. Immunohistochemical analysis of 5hmC and TET2 was performed in 133 BLCA specimens and 88 matched adjacent non-tumour tissues. TET2 mRNA expression was further evaluated using RT-qPCR and publicly available transcriptomic data from The Cancer Genome Atlas (TCGA) and genotype-tissue expression (GTEx) cohorts. Reduced 5hmC levels were significantly associated with muscle-invasive bladder cancer. Although high 5hmC expression was associated with a lower descriptive post-selection hazard, the survival association was not significant after accounting for the data-driven threshold search (Cutp p = 0.259). TET2 protein expression was significantly lower in BLCA than in adjacent non-tumour tissues. In contrast, both RT-qPCR and in silico analyses demonstrated increased TET2 mRNA expression in BLCA, revealing a previously unrecognised discordance between transcript and protein levels. This discrepancy may reflect post-transcriptional or post-translational regulation and/or tumour microenvironment-related effects. To the best of our knowledge, this is the first study to demonstrate reduced TET2 protein expression in BLCA using immunohistochemistry while simultaneously showing increased TET2 mRNA expression using independent transcriptomic approaches. Our findings suggest that 5hmC may have potential prognostic relevance in BLCA, and provide new insight into the complex regulation of TET2 in bladder tumorigenesis. The integrated assessment of TET2 at both the protein and transcript levels highlights the importance of multi-level molecular analyses for understanding epigenetic dysregulation in bladder cancer.