Upper urinary tract urothelial carcinoma (UTUC) is a poor-prognosis cancer of the renal pelvis and ureters. Due to its rarity, previous large-scale genomic studies of urothelial carcinomas have focused on bladder cancer, which is the commonest malignancy of the urinary tract. Recently, our study identified five molecular subtypes of UTUC with different outcomes. To further understand the molecular pathogenesis of UTUC, we performed whole-genome sequencing (WGS) of UTUCs.

We studied a total of 122 UTUCs which were surgically resected at the University of Tokyo hospital from 2000 to 2020. Matched germline control samples were also obtained from the normal renal cortex or peripheral blood samples. WGS was performed using NovaSeq at 120x and 30x depth for tumor and normal samples, respectively. G-CAT, GRIDSS, and FACETS were used to identify single nucleotide variants/insertion-deletions, structural variants (SVs), and copy number variants. Amplicon-suite was used to identify extrachromosomal DNA (ecDNA).

The median 11,000 mutations were detected per sample (range 2,700-780,000). Ten samples (8.1%) were hypermutated (>100,000 mutations), all of which had mutations in mismatch repair genes (MSH2, MSH6 and MLH1). dN/dS analysis identified 16 driver genes, of which the most frequently mutated driver gene was KMT2D in 54 samples (44.3%), followed by TP53 (33.6%), FGFR3 (32%), and ARID1A (23%). Recurrent non-coding mutations were also investigated, which identified known driver mutations in the promoter and 5' UTR regions of TERT. Comparing the frequency of mutations in the ureter and renal pelvis, TERT promoter and HRAS mutations were significantly more common in the ureter, while KMT2D and TP53 mutations were more frequently mutated in the renal pelvis.　Mutational signature analysis using SigProfiler identified SBS1 and SBS5, which are clock-like signatures and increase with age, and SBS2/13 caused by APOBEC mutagenesis. Mutations caused by APOBEC were identified in 87 cases and hotspot mutations in the non-coding regions accumulated on target motifs of APOBEC. SV analysis identified driver SVs, such as SVs involving FGFR3 (N = 10), which included 7 FGFR3-TACC3 translocations. In addition, KLF5 duplications and AHR deletions were observed in 9 and 7 samples, respectively. L1 retrotranspositions originating from TTC28 and PHACTR1 loci were also frequently detected as SVs. ecDNA was found in 58 samples (52%), including those containing oncogenes such as CCND1 and MDM2.

UTUCs were strongly affected by APOBEC mutagenesis, generating the hotspot mutations in non-coding regions. WGS identified non-coding driver mutations, driver SVs and ecDNAs, which have not been identified by the previous study.　These data could contribute to the refined UTUC classification and further understanding of its pathogenesis.