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.

Purpose: In NABUCCO, the safety and efficacy of preoperative ipilimumab plus nivolumab were assessed in stage III urothelial cancer. Encouraging responses were achieved, and ipilimumab 3 mg/kg (ipilimumab-high) seemed more effective than ipilimumab 1 mg/kg (ipilimumab-low). We explored ipilimumab plus nivolumab response biomarkers and tumor microenvironment (TME) treatment dynamics. Patients and Methods: Baseline formalin-fixed, paraffinembedded tumor tissue was analyzed using PD-L1 IHC (n ¼ 51) and whole-exome and transcriptome sequencing (both n ¼ 53) and correlated with response. Baseline infiltration of CD8⁺ T cells (n ¼ 51) and at cystectomy (n ¼ 42) was examined. Single-cell RNA sequencing (scRNA-seq) of CD3⁺ T cells was conducted on on-treatment resection tissue of two responders to ipilimumab-high to explore the characteristics of CD8⁺ T cells within the TME. Results: High tumor mutational burden and PD-L1 positivity were associated with response to ipilimumab plus nivolumab. Nonresponding patients exhibited increased expression of a TGFβ signature. We observed increased transcription of the g2m checkpoint and e2f target in responders to ipilimumab-high and enhanced transcription of IFN-α and IFN-γ hallmarks in responders to ipilimumab-low. CD8⁺TCF7⁺ T cells accumulated in the TME of responders to ipilimumab-high. scRNA-seq of CD8A⁺TCF7⁺ T cells demonstrated enhanced expression of IL7R, CCR7, GPR15, XCL1, SELL, and LEF1. Conclusions: Our data indicate that tumor mutational burden, PD-L1, and TGFβ are potential biomarkers for response to ipilimumab plus nivolumab in stage III urothelial cancer. An inflammatory TME might be relevant for responding to ipilimumab-low. We found that in responders to ipilimumabhigh, TCF7⁺CD8⁺ T cells accumulated in the TME. scRNA-seq in two responders suggested that TCF7⁺CD8A⁺ T cells express genes associated with immunologic memory formation and T-cell homing.

In prostate cancer, androgen receptor (AR)–targeting agents are very effective in various disease stages. However, therapy resistance inevitably occurs, and little is known about how tumor cells adapt to bypass AR suppression. Here, we performed integrative multiomics analyses on tissues isolated before and after 3 months of AR-targeting enzalutamide monotherapy from patients with high-risk prostate cancer enrolled in a neoadjuvant clinical trial. Transcriptomic analyses demonstrated that AR inhibition drove tumors toward a neuroendocrine-like disease state. Additionally, epigenomic profiling revealed massive enzalutamide-induced reprogramming of pioneer factor FOXA1 from inactive chromatin sites toward active cis-regulatory elements that dictate prosur-vival signals. Notably, treatment-induced FOXA1 sites were enriched for the circadian clock component ARNTL. Posttreatment ARNTL levels were associated with patients’ clinical outcomes, and ARNTL knockout strongly decreased prostate cancer cell growth. Our data highlight a remarkable cistromic plasticity of FOXA1 following AR-targeted therapy and revealed an acquired dependency on the circadian regulator ARNTL, a novel candidate therapeutic target. SIGNIFICANCE: Understanding how prostate cancers adapt to AR-targeted interventions is critical for identifying novel drug targets to improve the clinical management of treatment-resistant disease. Our study revealed an enzalutamide-induced epigenomic plasticity toward prosurvival signaling and uncovered the circadian regulator ARNTL as an acquired vulnerability after AR inhibition, presenting a novel lead for therapeutic development.