Detection of smoothelin expression in the urinary bladder is strongly dependent on pretreatment conditions: a critical analysis with possible consequences for cancer staging

Distinguishing urinary bladder muscularis propria (MP) from muscularis mucosae (MM) is crucial in bladder cancer staging. Immunohistochemical staining for the smooth muscle-specific protein smoothelin has been reported to be a robust marker for MP. The aim of this study was to investigate how smoothelin immunostaining in the bladder varies with pretreatment techniques and if it can be used to discriminate between MM and MP. Immunohistochemistry (IHC) for smoothelin was performed on nontumoral sections from 18 cystectomy specimens using three different pretreatment protocols. The immunoreactivity of MM, MP and blood vessels was scored semiquantitatively. Staining intensity depended strongly on the different pretreatment protocols used. Heat-induced epitope retrieval (HIER) in alkaline buffer resulted in the strongest staining with a moderate or strong immunostaining of the MP in 18/18 (100%) of cases, but in 11/18 (61%), the MM was moderately or strongly stained. HIER in acidic buffer resulted in a suboptimal staining of the MP. Enzymatic pretreatment resulted in absent or weak staining. In conclusion, smoothelin IHC is strongly dependent on epitope retrieval, and smoothelin staining did not discriminate reliably between MP and MM with any of the tested pretreatment protocols.