The Impact of Time-of-day Administration of EV/P on Objective Response Rate in Adults With Advanced Bladder Cancer

Objective response rate (ORR) will be determined by proportion of patients achieving a complete or partial response as assessed by RECIST v1.1 criteria, confirmed by central review or investigator assessment. Tumor assessments will be performed as standard of care (typically every 12 weeks), and best overall response prior to disease progression will be used for ORR determination.

Progression-free Survival (PFS) will be determined by time of randomization to radiographic progression or death from any cause, whichever occurs first.

Overall-survival (OS) is defined as the time from randomization to death from any cause. The analysis will compare OS curves between study arms and estimate 2-year OS rates for each group.

Time to treatment failure (TTF) is determined by assessing time from treatment initiation to treatment discontinuation for any reason, including disease progression, unacceptable toxicity, patient withdrawal, or death, capturing both treatment efficacy and tolerability.

Quantitative changes in circulating tumour DNA (ctDNA) levels will be collected and measured at baseline, post-cycle 1 (~3 weeks), and at post-cycle 4 (~12 weeks) at first radiographic assessment, with correlation to response and survival outcomes.

Differences in steroid use (yes/no) for the management of treatment-related toxicity, treatment interruptions (yes/no) due to treatment-related toxicity, and treatment discontinuations (yes/no) due to treatment-related toxicity will be used as surrogate safety outcomes, as comprehensive adverse event documentation is beyond the scope of this pragmatic clinical trial. The study seeks to determine whether treatment administration at a predefined time of day is associated with differences in the need for toxicity-related clinical interventions. Given that the relevant adverse events are well characterized in the existing literature, the focus of this study is on comparative safety between groups using these surrogate measures rather than on detailed characterization of individual adverse events.

Immune cell populations will be characterized from the blood samples at baseline (prior to treatment initiation) and at three predefined time points during therapy: 1 day after treatment initiation, 3 weeks into treatment, and 12 weeks into treatment. Cytometry by time-of-flight (CyTOF) will be used to comprehensively profile immune cell populations allowing for high-dimensional assessment of immune cell composition and activation states across treatment timing groups.

Bulk RNA sequencing (RNA-seq) will be performed to comprehensively characterize global gene expression profiles and to evaluate time-of-day-associated differences in immune-related transcriptional signatures. This approach will enable the identification of circadian variation in gene expression and provide insight into temporal regulation of immune pathways in response to treatment.

Plasma will be isolated for cytokine and chemokine analyses to assess systemic immune signaling and inflammatory profiles. Quantitative evaluation of circulating cytokines and chemokines will provide insight into treatment- and time-dependent changes in immune activation, inflammation, and immune regulation, complementing cellular and transcriptional immune profiling.

Circulating tumor DNA (ctDNA) analyses will be performed on plasma samples collected at baseline (prior to treatment initiation), 3 weeks into treatment, and 12 weeks into treatment. ctDNA dynamics will be evaluated to assess molecular response to therapy, including changes in ctDNA levels and ctDNA clearance rates over time. Comparisons will be made between treatment administration at different times of day to determine whether timing of therapy is associated with differences in ctDNA kinetics, clearance rates, and early molecular response.