Transcriptomics data from 11 historical cohorts (Kim80, Lindgren45, Sjödahl20127, CIT72, Choi81, Sjödahl201726, Song82, Sjödahl201983, Aarhus microarrays5,54,84,85,, Meeks35) were used for the validation of the four-class NMIBC classification and T1HG subtypes. The data were downloaded from GEO or ArrayExpress and annotated with HUGO Gene Symbols. In addition to using the publicly available data, we included data from three yet unpublished cohorts (listed below). Each sample in each cohort (n = 1228) was classified using the single-sample classifiers trained using the UROMOL cohort (1225 and 1226 tumors were assigned a class using the NMIBC classifier and T1HG classifier, respectively).

Data from Affymetrix Human Transcriptome 2.0 microarrays for 104 stage T1 and 113 stage Ta tumors from the Leeds Multidisciplinary Research Tissue Bank following approval by the research ethics committee 10/H1306/7). All ethical regulations for work with human participants were followed. Total RNA was isolated from frozen tissue sections using a RNeasy Plus Micro Kit and amplified using the Affymetrix GeneChip WT PLUS Reagent Kit. The resulting cDNA was hybridized onto Affymetrix Human Transcriptome 2.0 microarrays. Quality control checks, gene level normalization (using SST-RMA) and signal summarization was conducted using Affymetrix Expression Console Software.

RNA-Seq based analysis of 78 tumors from the West Midlands Bladder Cancer Prognosis Programme (BCPP, ethics approval 06/MRE04/65). All ethical regulations for work with human participants were followed. RNA libraries were prepared using the Truseq Stranded Total RNA with Ribo-zero Gold kit (Illumina) and 2 × 100 bp PE sequenced (Hiseq, n = 26) or 2 × 75 bp PE sequenced (Nextseq, n = 52). The data were aligned to GRCh37 and reads counted with STAR aligner (v2.5.2b). Log2(Read count+1) for each gene has been used as input for the class prediction.

RNA-Seq based analysis of 47 fresh frozen tumors from patients enrolled at Aarhus University Hospital with high-risk NMIBC, and analyzed following approval by the Danish National Committee on Health Research Ethics (#1708266). All ethical regulations for work with human participants were followed. RNA-Seq data was generated using analysis pipelines described above for the additional samples included in the discovery cohort in this work.

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