Background: Immunohistochemistry (IHC) plays a central role in subtyping of muscle-invasive bladder cancer (MIBC), yet conventional semi-quantitative scoring lacks objectivity and scalability. Automated digital pathology offers potential solutions, but requires robust, marker-specific validation against expert consensus scoring. Methods: We developed and internally validated an automated digital pathology pipeline for continuous IHC H-score quantification using QuPath (v0.6.0-arm64). Tissue microarrays (TMAs) were generated from transurethral resection of bladder tumor (TURBT) specimens from a cohort of patients with MIBC treated at Vancouver General Hospital. Cell detection was performed using StarDist (v0.9), followed by automated intensity-based H-score calculation for four epithelial IHC marker stains (CK14, CK20, CK5/6, and Uroplakin II). H-scoring was then restricted to tumor epithelium by object-level classification using a supervised tumor/non-tumor classifier trained on pathologist-reviewed annotations. Automated scores were compared with consensus scores from three blinded pathologists using Pearson correlation, linear regression, intraclass correlation coefficients (ICC), and Bland-Altman analysis. Results: Automated H-scores demonstrated strong agreement with pathologist consensus across all four markers. CK14 showed near-perfect agreement (ICC ≈ 0.99) with minimal bias and narrow limits of agreement. CK20 also showed high agreement (ICC ≈ 0.95). CK5/6 and Uroplakin II demonstrated slightly lower agreement (ICC ≈ 0.92 to 0.93) with mild proportional bias. Across markers, the automated pipeline preserved a broad H-score range, with range ratios of 0.96 to 0.99. Conclusions: This study establishes a robust, methods-forward pipeline for automated continuous IHC H-scoring in MIBC. The internally validated framework provides a scalable foundation for external cohort testing and future clinical outcome-associated biomarker analyses.
Diagnostics (Basel, Switzerland). 2026 May 29*** epublish ***
Matthew Yap, Ioana-Maria Mihai, Maram Awadh A Alanazi, Gheorghe-Emilian Olteanu, Alberto Contreras-Sanz, Peter Black, Gang Wang
Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC V6T 1Z7, Canada., Pathology and Laboratory Medicine, BC Cancer, Vancouver, BC V5Z 4E6, Canada., Department of Urologic Sciences, University of British Columbia, Vancouver, BC V6H 3Z6, Canada.