SEPT9 methylation as a potential biomarker for uncovering latent malignancy in flat urothelial lesions

Accurate differentiation between reactive and neoplastic flat urothelial lesions is critical for patient management; however, cystoscopic biopsy often results in false negatives because of sampling limitations and interpretive challenges. This study evaluated the diagnostic utility of SEPT9 methylation as a molecular biomarker in 134 cystoscopic biopsy lesions, categorized as reactive urothelial atypia (RUA, n = 58), dysplasia (DYS, n = 25), carcinoma in situ (CIS, n = 12) and indeterminate lesions (n = 39). SEPT9 methylation levels showed a stepwise increase from RUA to indeterminate lesions and further to DYS and CIS (p < 0.0001), suggesting involvement in early urothelial carcinogenesis. Methylation positivity was detected in 6.90% (4/58) of patients with RUA and 91.89% (34/37) of patients with neoplastic lesions (DYS and CIS), supporting its diagnostic potential. Among lesions with initially indeterminate histology, 88.24% (15/17) of SEPT9-positive cases were later confirmed to be urothelial carcinoma (UC), compared to only 4.55% (1/22) of negative cases. These findings indicate that SEPT9 methylation is a promising adjunct to histopathology that can distinguish benign from neoplastic lesions and uncover latent malignancies in morphologically ambiguous flat urothelial lesions.