Background: Active Wnt signaling via WNT5A through ROR1 and its partner, ROR2, or WNT5A/frizzled 2 (FZD2) is linked to processes driving tumorigenesis, disease progression and therapy resistance. The role of this pathway in the pathogenesis of urothelial carcinoma (UC) is not fully elucidated. In adult tissue, ROR1 is largely absent, which makes it ideal for targeted therapies, with several ROR1 targeting agents in early clinical development. We utilized a large dataset of molecularly characterized UC tumors to investigate the significance of Wnt5a/ROR1, ROR2 or FZD2 transcriptional expression.

Methods:NextGen Sequencing of DNA (592 genes or WES)/RNA (WTS) was performed for4743 UC tumors submitted to Caris Life Sciences (Phoenix, AZ). PD-L1 expression (SP142; Positive (+): ³2+, ³%5) was tested by IHC. Gene expression profiles were analyzed for a transcriptional signature predictive of response to immunotherapy (T cell-inflamed; Bao, 2020). WNT5a, ROR1, ROR2, and FZD2-high and -low expression were defined as ³ top and < bottom quartile of transcripts per million (TPM), respectively. Mann-Whitney U and X²/Fisher-Exact tests were applied where appropriate, with P-values adjusted for multiple comparisons (q < .05). Real-world overall survival (OS) information was obtained from insurance claims data and Kaplan Meier estimates were calculated for molecularly defined cohorts.

Results: We observed similar expression of these WNT5A signaling pathway genes between upper (N=795) and lower tract UC (N=3,204): WNT5A (22.7 v. 22.2 median TMP (mTPM), q = .18), FZD2 (3.4 v 3.5, q = .93), ROR1 (2.0 v. 1.7, q = .05), and ROR2 (2.1 v 2.5, q < .01). WNT family gene expression varied significantly between primary (N=2,756) and metastatic sites (N=1,361): WTN5A (25.2 v 16.8 mTPM), FZD2 (3.2 v 4.05), ROR1 (1.7 v 2.1), and ROR2 (2.4 v 2.6) for primary vs. metastatic sites respectively (q < .05 for all). Comparison of high- and low-expression subgroups revealed variation in the prevalence of TP53, FGFR3 and RB1 mutations, as well as PDL1+ staining and T cell-inflamed scores (Table). High gene expression for ROR2 (HR 0.66, 95% CI 0.56-0.78, p < .001) and FZD2 (HR 0.75, 95% CI 0.63-0.89, p < .001) was associated with worse OS compared to low gene expression. No significant difference in OS was observed for WNT5A (HR 0.97, 95% CI 0.82-1.15, p < .76)and ROR1 (HR 0.86, 95% CI 0.72-1.01, p < .068).

Conclusions: Distinct genomic and immune landscapes for the four investigated WNT pathway components were observed and should be leveraged to identify combination therapies that complement the current pipeline of WNT pathway-targeting drugs.