Urinary DNA Methylation Profiling Identifies a Biologically Distinct Subgroup of Low-Grade Urothelial Carcinoma with High-Grade-Associated Features

Bladder cancer surveillance remains challenging because conventional methods do not fully capture the biological heterogeneity within histologically low-grade urothelial carcinoma (LGUC). We evaluated whether urinary DNA methylation profiling using Bladder EpiCheck™ can identify clinically relevant LGUC subgroups in a retrospective single-center cohort of 134 patients with paired histopathology, urine cytology, and methylation results. Methylation positivity increased across negative histopathology, LGUC, and high-grade urothelial carcinoma (HGUC) (18.4%, 51.4%, and 91.5%, respectively). Within LGUC, 19 of 37 tumors (51.4%) were methylation-positive. Their EpiScores overlapped those of HGUC, whereas methylation-negative LGUCs exhibited scores similar to negative histopathology. Gaussian mixture modeling independently supported a bimodal distribution. Methylation-positive LGUCs demonstrated a higher Ki67 labeling index, more frequent MTAP loss (33.3% vs. 11.8%), and more recurrences, including recurrence as HGUC, than methylation-negative LGUCs. Conventional nuclear morphometry did not distinguish between the two LGUC subgroups. These exploratory findings suggest that urinary DNA methylation may identify a biologically distinct subset of histologically low-grade tumors with selected high-grade-associated features. Prospective external validation is required before methylation status can inform surveillance or treatment decisions.