Pathway enrichment analysis was performed independently in the UROMOL cohort and each historical cohort that contained representatives of all classes. First, we collected pathway annotation from the Reactome (using R package reactome.db v1.68.0) and KEGG (using the R package KEGGREST v1.24.1) databases. We joined these annotations and performed gene-set variation analyses (using the R package GSVA v1.32.0) to obtain single-sample enrichment scores for each pathway.

To find associations between pathways and classes, we performed Mann–Whitney U-tests using the pathway enrichment scores between samples in each class versus samples in other classes in each cohort separately. P-values were BH-adjusted. For the pathway visualizations, we first filtered pathways that were enriched in the same class in the UROMOL cohort and in at least four other datasets and then manually selected pathways from the filtered list. Pathway enrichment scores were grouped using hierarchical clustering with correlation distances (1 – r) and Ward clustering using the enrichment scores in the UROMOL cohort and the same pathway order was then used for the independent cohorts.

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