Background: Pre-operative immune checkpoint blockade (ICB) with ipilimumab and nivolumab has shown encouraging pathological complete response (pCR) rates in stage III urothelial cancer (UC). A previous analysis of NABUCCO suggested that ipilimumab 3 mg/kg is more effective than ipilimumab 1 mg/kg. However, long-term progression-free and overall survival (PFS, OS) following pre-operative combination ICB are unknown. Methods: In NABUCCO, 54 patients received pre-operative ipilimumab plus nivolumab in different dosing regimens. PFS and OS were determined for the entire NABUCCO population and various clinically relevant subgroups. We explored ICB effects on the cellular composition of tumor-draining lymph nodes (tdLN) from ICB-treated patients (n = 5) and untreated or chemotherapy-treated patients (n = 5) using multiplex immunofluorescence for the PhenoCycler Fusion (Akoya). Results: With a median follow-up of 70 months, PFS and OS at 60 months were 67 % and 70 %, respectively, for the entire study. PFS and OS at 60 months were similar for patients with residual non-muscle invasive UC (NMIBC) and patients with a pCR. The presence of a nodal micrometastasis (<2 mm) after ICB, the development of grade ≥ 3 immune-related adverse events (irAE) and corticosteroids or antibiotics did not negatively impact survival. We observed smaller distances from CD20⁺ cells to CD14⁺ cells in tdLN following ICB compared to tdLN from untreated or chemotherapy-treated patients. Conclusions: Our data demonstrate a 5-year PFS of 67 % and OS of 70 % after pre-operative ICB in stage III UC. Survival was not impaired for patients with residual NMIBC, a nodal micrometastasis at resection, grade ≥ 3 irAE or corticosteroid use.

Immune checkpoint inhibitors (ICI) can achieve remarkable responses in urothelial cancer (UC), which may depend on tumor microenvironment (TME) characteristics. However, the relationship between the TME, usually characterized by immune cell density, and response to ICI is unclear. Here, we quantify the TME immune cell densities and spatial relationships (SRs) of 24 baseline UC samples, obtained before pre-operative combination ICI treatment, using multiplex immunofluorescence. We describe SRs by approximating the first nearest-neighbor distance distribution with a Weibull distribution and evaluate the association between TME metrics and ipilimumab+nivolumab response. Immune cell density does not discriminate between response groups. However, the Weibull SR metrics of CD8⁺ T cells or macrophages to their closest cancer cell positively associate with response. CD8⁺ T cells close to B cells are characteristic of non-response. We validate our SR response associations in a combination ICI cohort of head and neck tumors. Our data confirm that SRs, in contrast to density metrics, are strong biomarkers of response to pre-operative combination ICIs.

Candidate immune biomarkers have been proposed for predicting response to immunotherapy in urothelial cancer (UC). Yet, these biomarkers are imperfect and lack predictive power. A comprehensive overview of the tumor immune contexture, including Tertiary Lymphoid structures (TLS), is needed to better understand the immunotherapy response in UC. We analyzed tumor sections by quantitative multiplex immunofluorescence to characterize immune cell subsets in various tumor compartments in tumors without pretreatment and tumors exposed to preoperative anti-PD1/CTLA-4 checkpoint inhibitors (NABUCCO trial). Pronounced immune cell presence was found in UC invasive margins compared to tumor and stroma regions. CD8⁺PD1⁺ T-cells were present in UC, particularly following immunotherapy. The cellular composition of TLS was assessed by multiplex immunofluorescence (CD3, CD8, FoxP3, CD68, CD20, PanCK, DAPI) to explore specific TLS clusters based on varying immune subset densities. Using a k-means clustering algorithm, we found five distinct cellular composition clusters. Tumors unresponsive to anti-PD-1/CTLA-4 immunotherapy showed enrichment of a FoxP3⁺ T-cell-low TLS cluster after treatment. Additionally, cluster 5 (macrophage low) TLS were significantly higher after pre-operative immunotherapy, compared to untreated tumors. We also compared the immune cell composition and maturation stages between superficial (submucosal) and deeper TLS, revealing that superficial TLS had more pronounced T-helper cells and enrichment of early TLS than TLS located in deeper tissue. Furthermore, superficial TLS displayed a lower fraction of secondary follicle like TLS than deeper TLS. Taken together, our results provide a detailed quantitative overview of the tumor immune landscape in UC, which can provide a basis for further studies.