Epigenomic analysis of high-grade T1 bladder cancer reveals subtypes with clinical implications that display extensive intra-tumor heterogeneity with spatial features.

Abstract High-Grade T1 (HGT1) Non-Muscle Invasive is an early-stage of Bladder Cancer (NMIBC) with an unpredictable relapse and recurrence rate. New biomarkers are needed for patient risk prediction and to determine treatment. Here, we analyze the molecular characteristics of HGT1 NMIBC by combining multi-omics at bulk and single-cell resolution. We have identified two chromatin states that reflect variable degrees of basal and luminal inflammatory characteristics. These subtypes show relevant functional differences and are associated with clinical outcome. Luminal-like inflammatory (LLI) tumors are associated with a significantly higher risk of progression. At a single-cell resolution, RNA-seq shows the existence of a high degree of intra-tumor heterogeneity with co-existence of LLI and Basal-like (BL) subtypes within the same tumor specimen. The immunohistochemical validation in an independent patient cohort reveals subtypes coexistence in more than 40% patients. The subtypes show distinct spatial features within the tissue with the BL subtype consistently being in close proximity to the vascular stroma and the LLI subtype towards the interior of the tumor mass. Spatial transcriptomics at a single cell resolution further validate the specific location of the subtypes. Altogether, our results illustrate the high intratumor heterogeneity existing in HGT1 NMIBC bladder cancer, with spatial features that suggest microenvironment crosstalk. Based on our findings, multitarget therapy could be needed to target the extensive intra-tumor heterogeneity existing in bladder cancer.