Intrinsic molecular subtypes with luminal or basal features have been identified in both breast and bladder cancer as promising areas for biomarker development. These intrinsic molecular subtypes may explain marked variation in prognosis and response to therapy. However, complex testing algorithms and little attention to more prevalent, early stage, non-muscle-invasive bladder cancers (NMIBCs) have hindered implementation of subtyping in routine clinical practice. Furthermore, the current grading classification for NMIBC includes a low-risk category known as PUNLMP and has been understudied with respect to molecular subtypes that could clarify its diagnostic distinction from other low-grade and high-grade NMIBCs. It is unclear whether subtype-specific clinical behaviors arise from tumour-intrinsic genomic features or signals from the surrounding benign tissue (stroma). We propose that profiling tumour and stromal immune cell populations across NMIBC samples may identify features contributing to the variability in prognoses and sensitivities to intravesical BCG immunotherapy. Current research suggests that clinical outcomes may be attributed to differences in the tumour immune microenvironment (TIME) of NMIBC patients, including immune cell populations such as CD3+/CD8+ T-cells, M2 macrophages and B-cells in the tumour and stromal compartments. However, simultaneous profiling of the TIME coupled with patient outcomes have not yet been described in NMIBC. To address these issues, we have developed a simpler, more robust method for molecular subtyping of NMIBC. We identified protein assays routinely used in surgical pathology laboratories that represent cardinal features of luminal and basal subtypes. Here, we validate the diagnostic and prognostic significance of molecular subtypes in PUNLMP and NMIBC cohorts using a simplified three-antibody algorithm. Furthermore, we used multiplex IHC methods to comprehensively profile both immune cell populations and immune checkpoints using three separate IHC panels for T-cells (CD3, CD8, Ki67, FOXP3), B-cell/Macrophage markers (CD103, CD163, CD79a) and PD1/PD-L1 (PD1, PD-L1, IDO1, CK5). Applying these profiling methods to NMIBC, we explore the differences in tumour and stromal immune microenvironments and their associations with prognosis. This work examines the prognostic associations between molecular subtypes across NMIBC tumour grades in conjunction with their immune microenvironment, and in doing so, reveals key biological and clinical differences amongst NMIBC tumours.
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