4571 Background: Enfortumab vedotin improves response and outcomes over chemotherapy in mUC, with further benefit when combined with pembrolizumab. Although EV alone has limited immune-activating effects, we hypothesize that the combination of EV+P modulates peripheral T-cell activation states and correlates with clinical response. Methods: We conducted a retrospective analysis of mUC patients treated with EV+P at Memorial Sloan Kettering Cancer Center. PBMCs were isolated from patients on C1D1 and C2D1 of EV+P (n=33) and analyzed with a 32-color spectral flow cytometry panel. Immune subsets were compared between responders (complete [CR] or partial response [PR]) and non-responders (stable [SD] or progressive disease [PD]) using Wilcoxon rank sum test. Response was determined by a radiologist’s assessment of the best-overall response on EV+P according to RECIST v1.1. Results: Among 33 patients, median age was 71 years and 48% were male. 12.1% (4/33) received prior platinum chemotherapy and 18.1% (6/33) received prior PD-1/PD-L1 immunotherapy. The objective response rate was 67% (CR=10; PR=12); eight patients had SD and three had PD. At C1D1, CD8+ T cell frequencies did not differ by response. At C2D1 however, circulating CD8+ T cells were significantly higher in non-responders (p < 0.05). From baseline to C2D1, CD8+ T cells showed increased expression of CTLA4+ (p < 0.0001), Ki67+ (p < 0.01) and HLA-DR+ (p < 0.01), with higher expression of each marker at C2D1 significantly correlated with response (all p < 0.05, Table 1). At C2D1, CD4+ effector T cells showed increased expression of Ki67+ and HLA-DR+ (both p < 0.05), both significantly higher in responders (both p < 0.05). CD4+ regulatory T cells showed increased CTLA4+ (p < 0.001), Ki67+ (p < 0.01) and HLA-DR+ (p < 0.01) expression from C1D1 to C2D1, with significantly higher expression in responders (CTLA4+ p < 0.05, Ki67+ p < 0.0001, HLA-DR+ p < 0.001). Across all T cell subtypes, PD1+ expression significantly decreased (p < 0.05) from C1D1 to C2D1, but this decrease did not correlate with response. The frequencies of circulating memory CD8+ and CD4+ T cells did not change on treatment or correlate with response at either timepoint. Conclusions: EV+P induces early peripheral T cell activation in mUC, with increased expression of activation and proliferative markers in responders at C2D1. Ongoing studies are investigating correlation between TCR specificity, immune activation and responses to EV+P. Peripheral CD8+ T cell frequencies (median) at C1D1 and C2D1 from mUC patients on EV+P. CD8+ T Cell C1D1 C2D1 p-value C2D1 in Responders C2D1 in Non-Responders p-value CD8+ (%CD45+) 8.54 7.8 0.6 7.27 13.8 0.03 CTLA4+ (%CD8+) 1.15 3.94 0.0001 4.57 2.85 0.02 Ki67+ (%CD8+) 3.18 6.27 0.005 7.26 5.4 0.04 HLA-DR+ (%CD8+) 4.66 9.37 0.004 10.22 4.13 0.04 PD-1+ (%CD8+) 45.6 29.8 0.0005 28.7 31.5 0.48

4514 Background: Maintenance avelumab is a standard of care in patients (pts) with la/mUC who do not progress after first-line PBC. Cabozantinib (CABO) is an oral inhibitor of MET, VEGFR and TAM, active in multiple solid tumors. We hypothesized that an avelumab (Av)-CABO combination would improve outcomes in pts with mUC with an acceptable safety profile compared to avelumab maintenance alone. Methods: MAIN-CAV (NCT05092958) is a phase III randomized, multicenter, international trial for la/mUC pts who did not progress after 4-6 cycles of any PBC (gemcitabine-cisplatin, gemcitabine-carboplatin, MVAC, ddMVAC). Pts were randomized 1:1 to Av 800 mg IV every 2 weeks alone or with CABO 40 mg daily for ≤2 years. Stratification factors were response to first-line PBC (complete response [CR] vs partial response [PR] vs stable disease [SD]) and visceral metastases. The primary endpoint was overall survival (OS); secondary endpoints included progression-free survival (PFS), safety and quality of life. The target accrual was 654 pts; the trial closed early after enrollment of 68 pts following adoption of enfortumab vedotin plus pembrolizumab as the new preferred first-line standard in the US. Results: From 3/2022 to 3/2024, 68 pts were randomized (Av n = 33; CABO-Av n = 35). Median OS was 24.8 months (mo) with Av and 23.0 mo with Avb-CABO (Hazard ratio 1.41; 95% CI 0.66–3.01; p = 0.186). Median PFS was 4.6 mo with Av vs 6.0 mo with Av-CABO (p = 0.343). Objective response rate among pts with PR or SD after first-line PBC was 20.8% with Av vs 11.5% with Av-CABO (p = 0.424). Grade ≥3 treatment-related adverse events occurred in 24.2% pts on the Av arm vs 39.4% of pts on the Av-CABO arm and CABO dose reductions occurred in 63% of pts. Conclusions: Addition of CABO to Av did not improve OS or PFS compared with Av alone in this underpowered, prematurely closed phase III trial; safety was consistent with known profiles. U10CA180821,U10CA180882; U24CA196171, U10CA180863. (CCTG);U10CA180888 (SWOG); https://acknowledgments.alliancefound.org. Clinical trial information: NCT05092958 .

4500 Background: BM occur in ~30% of RCC pts and are associated with poor survival and symptomatic skeletal events (SSEs). Radium-223, an alpha-emitting bone-seeking radioisotope, prolongs survival in advanced prostate cancer. A pilot study of radium-223 with VEGF tyrosine kinase inhibitors (TKI) in RCC with BM showed safety and early efficacy (McKay, CCR, 2018). Cabozantinib is a TKI that showed efficacy in BM. RADICAL (NCT04071223) was designed to investigate cabozantinib ± radium-223 in RCC with BM. Methods: This open label multicenter study enrolled RCC pts with ≥1 symptomatic BM not previously irradiated and Karnofsky performance status ≥60%. Prior therapies were allowed; non-clear cell population was limited to 20%. Randomization was 1:1 to cabozantinib with (Arm A) or without (Arm B) radium-223, stratified by osteoclast-targeted therapy (OTT), prior therapy, baseline opioid use, and IMDC risk. In Arm A, cabozantinib was 40 mg daily cycle 1, 60 mg thereafter if tolerated; radium-223 was 1.49 microcurie/kg IV every 28 days for 6 doses. In Arm B, cabozantinib was 60 mg daily. The primary endpoint was SSE-free survival (SSE-FS). Secondary endpoints were safety, objective response rate (ORR), progression-free survival (PFS) and overall survival (OS). The study was designed with 85% power to detect improvement in 6-month SSE-FS from 65% to 78% (one-sided α=0.05), with planned interim futility analysis at 50% of expected events. Results: From 12/2019 to 9/2025, 90 pts were enrolled and evaluable for the interim analysis. Median age was 63 years; 74.4% male, 90.0% White, 83.3% clear cell histology. 11.1% were treatment naïve, 80.0% received OTT, 51.1% had baseline opioid use. IMDC risk was 16.7% favorable, 68.9% intermediate, 14.4% poor. Median cabozantinib dose was 31.4 mg (Arm A) and 40.0 mg (Arm B); median radium-223 cycles was 6. Median follow-up was 13.1 (range 0.1-49.4) months with 50 events, 17 SSEs (Arm A: 10; Arm B: 7) and 33 deaths (Arm A: 13; Arm B: 20). Outcomes are in Table. 71 pts were evaluable for response. ORR was 22.2% (Arm A), 22.9% (Arm B). Any grade treatment-related adverse events were 97.7% (Arm A), 93.2% (Arm B); grade ≥3 were 65.9% (Arm A), 56.8% (Arm B). The trial closed after interim analysis as SSE-FS did not meet the prespecified futility boundary (HR≤1). Conclusions: Radium-223 did not improve the primary endpoint of SSE-FS when added to cabozantinib but a numerical prolongation in OS was observed. Combination treatment was safe. Alternative radiotherapeutics warrant investigation in RCC. Clinical trial information: NCT04071223 . Interim efficacy analysis. Arm Median(95% CI) HR (95% CI) Stratified SSE-FS A 17.9 (15.0-NE) 1.24 (0.62-2.48) B 17.6 (10.9-NE) Unstratified SSE-FS A 17.9 (15.0-NE) 0.90 (0.51-1.59) B 17.6 (10.9-NE) OS A 32.2 (17.9-NE) 0.77 (0.42-1.41) B 21.3 (12.3-NE) Stratified PFS A 11.0 (5.8-17.4) 1.39 (0.72-2.66) B 11.2 (9.0-18.5)

Abstract Background Diet and metabolism are increasingly recognized as modifiable determinants of response to immune checkpoint blockade (ICB), yet the impact of specific metabolites in metastatic renal cell carcinoma (mRCC) remains poorly defined. Preclinical data in melanoma link fructose consumption to ICB resistance. In our prior work, however, higher dietary fructose showed a univariable association with longer overall survival (OS) in a small RCC cohort. The fructose-metabolism enzyme ketohexokinase (KHK) is downregulated in clear cell RCC cells, raising the possibility that tumor-level fructose handling is biologically relevant. We evaluated whether dietary fructose intake and tumoral expression of fructose-metabolism genes are associated with outcomes in patients with mRCC receiving ICB. Methods We conducted a retrospective analysis of a prospectively collected cohort of patients with mRCC treated with ICB. Dietary intake was assessed using the validated Harvard Willett Food Frequency Questionnaire. Fructose intake was modeled as a continuous variable in raw (g/d) and energy-adjusted (g/d per 1,000 kcal) form. Associations with progression-free survival (PFS) and OS were evaluated by univariable (UV) and multivariable (MV) Cox proportional hazards regression, adjusted for International Metastatic RCC Database Consortium (IMDC) risk, histology, and treatment regimen. Kaplan-Meier curves were used to visualize associations of fructose intake and survival outcomes. To assess biologic plausibility, tumoral expression of fructose-metabolism enzymes, specifically KHK, was examined in the TCGA Kidney Renal Clear Cell Carcinoma (KIRC) PanCancer Atlas and in IMmotion151, CheckMate 214, and JAVELIN Renal 101. Results Sixty-eight patients with mRCC were included. Median age was 64 years, 74% were male, and 18% had non-clear cell histology; 57% received an ICB+TKI regimen and 43% a dual ICB regimen. Median dietary fructose intake was 17.0 g/d (10.2 g/d per 1,000 kcal). Fructose intake was not correlated with IMDC risk score, ECOG performance status, or BMI. Higher fructose intake was significantly associated with longer OS in both UV (log-rank p < 0.001) and MV analyses: each 10 g/d increase in raw fructose was associated with an adjusted hazard ratio (aHR) for OS of 0.47 (95% CI 0.27–0.83, p = 0.009), with a directionally concordant energy-adjusted estimate (aHR 0.37 per 10 g/d per 1,000 kcal, 95% CI 0.14–0.98, p = 0.047). No association was observed with PFS in either model. At the tumor level, higher KHK expression trended towards association with longer survival across all evaluable RCC datasets, reaching statistical significance in the TCGA-KIRC (OS HR 0.70, 95% CI 0.58–0.85, p < 0.001), both arms IMmotion151 (atezolizumab + bevacizumab arm, PFS HR 0.85, 95% CI 0.75–0.96, p = 0.007; sunitinib arm, PFS HR 0.70, 95% CI 0.63-0.79, p < 0.001) and the sunitinib arm of JAVELIN Renal 101 (0.65, 95% CI 0.56-0.75, p < 0.001). Effect sizes were similar or greater in TKI-containing arms, suggesting a predominantly prognostic rather than ICB-specific role. Conclusions In our mRCC cohort treated with ICB-based therapy, higher dietary fructose intake was associated with longer OS. Concordantly, higher tumoral KHK expression was associated with improved survival outcomes across multiple public RCC datasets, suggesting a biological relationship between fructose metabolism and favorable clinical outcomes in RCC. Whether RCC tumors with active fructose metabolism may represent a less aggressive disease phenotype warrants further investigation. Mechanistic studies linking dietary fructose to RCC tumor biology are ongoing and may inform dietary strategies in mRCC.

4517 Background: Enfortumab vedotin plus pembrolizumab (EVP) is the current first-line standard of care for locally advanced / metastatic urothelial carcinoma (mUC). However, subsequent treatment options are limited, representing an urgent and growing unmet need. Preclinical data suggest that EV resistance may be payload mediated and consequently Nectin-4 remains a viable target for alternative therapies. LY4101174 is a next generation anti-Nectin-4 ADC comprising a humanized IgG1 antibody conjugated to the topoisomerase I inhibitor, exatecan, via a maleimide-ß-glucuronide poly-sarcosine linker and a homogeneous drug antibody ratio of 8. Here, we report the initial clinical data from the phase 1 dose escalation cohort of EXCEED (NCT06238479). Methods: Adults with locally advanced / mUC or other selected solid tumors were eligible. Participants (pts) must have received or were ineligible for available standard therapies, and have ECOG PS 0-1. Dose escalation utilized a Bayesian optimal interval design. Key endpoints were safety, PK, and antitumor activity of LY4101174 per RECIST v1.1. Results: As of 3 Dec 2025, 143 pts (86 mUC, 57 non-UC tumors) were treated across 6 dose levels (DLs) of LY4101174 (0.8-3.2 mg/kg Q2W or 2.4-4.0 mg/kg Q3W IV). Median age was 68 (range, 30-85), 60% ECOG PS 1. Median prior lines of therapy was 4 (range, 1-10), 92% of mUC pts had received prior EV (19% [15/79] had discontinued EV due to treatment-related toxicity). LY4101174 and total antibody exhibited mostly linear, dose-proportional PK up to 4.0 mg/kg, and up to 3.2 mg for exatecan. At 3.2 mg/kg Q2W, 3 of 6 pts experienced DLTs (neutropenia [grades 3 and 4], febrile neutropenia, and thrombocytopenia [both grade 3]) prompting a schedule change to 3.2 mg/kg Q3W for continued escalation. The most common treatment-emergent AEs (TEAEs) across Q3W DLs were anemia (71%), fatigue (55%), nausea (49%), neutropenia (36%), thrombocytopenia (31%), diarrhea (26%), vomiting (25%), and decreased appetite (23%). The most common grade ≥3 TEAEs were anemia (47%), neutropenia (29%), thrombocytopenia (21%), febrile neutropenia and leukopenia (10% each); prophylactic granulocyte colony stimulating factor (G-CSF) was required at DL 4.0 mg/kg Q3W. TRAEs led to dose reduction in 24% and discontinuation in 2%. In the 66 efficacy evaluable mUC pts treated with 2.4-4.0 mg/kg Q3W (92% [61/66] EV-pretreated), ORR was 18% (12/66) and DCR was 70% (46/66) with 12 PR (8 confirmed, 4 ongoing and pending confirmation) and 34 SD. Median follow-up was 4.9 months (95% CI, 1.4-NE). Conclusions: LY4101174 has demonstrated clinical activity at Q3W dose levels, including in EV pre-treated mUC, suggesting Nectin-4 remains an important therapeutic target. Dose and duration on treatment were mainly limited by grade ≥3 hematologic toxicity requiring the use of prophylactic G-CSF. Clinical trial information: NCT06238479 .

Abstract EP300 and CREBBP encode p300 and CBP respectively, which are H3K27 acetylases. Both EP300 and CREBBP mutated in a subset of urothelial cancers (∼15% for EP300 or CREBBP). The EP300 and CREBBP genes have ∼60% sequence similarity and thus have been presumed to have largely overlapping functional roles in cell homeostasis and cancer pathogenesis. To characterize the role of EP300 mutations in urothelial cancer pathogenesis and to identify non-redundant roles of these paralogues, we generated EP300 and CREBBP isogenic knockout urothelial cancer cell lines and characterized low passage mutant and wild type for EP300 and CREBBP urothelial cancer-derived patient derived organoids. EP300 KO and loss-of-function mutation was associated with enhanced cell growth in soft agar, increased invasive potential in vitro and altered cellular metabolism. These gain-of-function phenotypes were mediated by enhanced JAK-STAT3 activation resulting from IL-6 trans-signaling, the proximal driver of which was increased transcription and production of IL-1á. Notably, isogenic BLCA cells with CREBBP knockout did not confer IL-1á hypersecretion or hyperactivation of the IL-6/JAK1/STAT3 signaling axis indicating that this is a phenomenon was specific to EP300 loss-of-function. Transcriptomic analysis of Parental, EP300 KO, and CREBBP KO RT112 clones revealed that CREBBP KO significantly depressed IL1A transcript levels which EP300 KO significantly elevated. Additionally, CREBBP inactivated BLCA lines could not upregulate IL1A expression following genotoxic stress in contrast to parental and EP300 null cell lines. Using an inducible short hairpin RNA construct targeting CREBBP, we also find that CREBBP knockdown rescued IL1A upregulation in EP300 ko clones that coincided with a significant growth defect. Pharmacologic inhibition via the p300/CBP specific inhibitor A485 also abolished IL1A upregulation and caused significant growth defect in EP300 KO cells. EP300 null clones were also significantly more sensitive to A485 treatment than either CREBBP null urothelial cell or parental cell lines. In sum, our results identify regulation of IL-1á-JAK-STAT3 signaling as a novel non-redundancy between EP300 and CREBBP that could be exploited therapeutically in patient with EP300 loss-of-function mutations. Citation Format: James August Rodrigues, Hikmat A. Al-Ahmadie, Sizhi P. Gao, Jiaqian Luo, Jacob Tallman, Fengshen Kuo, Merve Basar, Cansu Yol, Jordan Eichholz, Alejandra Lopez Rojas, Ecenur Turkay, Jonathan E. Rosenberg, Gopa Iyer, Eugene J. Pietzak, Dan Li, Dana Schoeps, Shipra Shukla, Zoe Jacobs, Chen Khuan Wong, Woo Hyun Cho, Ping Chi, David B. Solit, Yu Chen, . CREBBP drives tumorigenicity via aberrant IL-1α signaling in EP300 altered bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4757.

Abstract Background: Dietary fiber intake has been associated with longer PFS on ICB in observational studies, putatively due to effects of fiber on the gut microbiome. However, associations in observational studies may be due to confounding rather than causal relationships. Dietary animal protein intake impacts health and can be correlated with fiber intake. We investigated whether animal protein intake is associated with ICB outcomes in UC and RCC and whether confounding by animal protein intake might account for associations of fiber intake with ICB outcomes. Methods: We conducted a retrospective analysis of a prospectively collected cohort, wherein baseline dietary data were collected using the Willett Food Frequency Questionnaire from pts with advanced UC or RCC initiating ICB at Memorial Sloan Kettering. Associations of diet with clinical outcomes were assessed using univariable and multivariable Cox proportional hazards regression. Results: From 2/2021-6/2022, 86 pts eligible for this analysis were enrolled (UC n = 38; RCC n = 48; median follow-up 14.6 months). Median animal protein intake was 44 g/day (interquartile range [IQR] 31-54), below the American average of 88 g/day. Median fiber intake was 17.2 g/day (IQR 13-24), similar to the American average of 15 g/day. Intake of animal protein and fiber were correlated (Spearman’s rho 0.46, p < 0.001). There were no statistically significant associations of animal protein intake above vs below the median with PFS in either univariable or multivariable models (Table 1). Dietary fiber intake was associated with longer PFS even after adjusting for animal protein intake and clinical covariables (Table 1). Conclusion: Dietary animal protein intake was not associated with PFS among pts with UC and RCC on ICB. The association of dietary fiber with longer PFS could not be attributed to confounding by animal protein intake. Citation Format: Timothy B. Winslow, Ritesh Kotecha, Neha Ratna, David Gavrilov, Cihan Duzgol, Mahnoor Akhlaq, Min Yuen Teo, Ashley M. Regazzi, Samuel Funt, David H. Aggen, Scot Niglio, Maria Carlo, Daniel Lage, Neil Shah, Aditi Gupta, Darren F. Feldman, Martin H. Voss, Robert J. Motzer, Jonathan E. Rosenberg, Gopa Iyer, Brendan John Guercio. Dietary animal protein intake and progression-free survival (PFS) of patients (pts) with advanced urothelial cancer (UC) and renal cell carcinoma (RCC) on immune checkpoint blockade (ICB) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2862.

PURPOSE:Many patients with muscle-invasive bladder cancer (MIBC) are ineligible for cisplatin-based therapy. We conducted a phase II trial of neoadjuvant nivolumab ± ipilimumab for cisplatin-ineligible patients. PATIENTS AND METHODS:Patients with MIBC were enrolled in two consecutive cohorts: (i) nivolumab alone and (ii) ipilimumab/nivolumab. A third cohort with alternative dosing was planned. The primary endpoint was eligibility for cystectomy ≤60 days after last treatment. Correlative analyses were performed, with the PURE-01 trial used as an independent dataset. RESULTS:Fifteen patients enrolled onto each cohort. In cohorts 1 and 2, 12 of 15 and eight of 15 were eligible for cystectomy within 60 days, respectively. Due to cohort 2's failure to meet the primary endpoint, cohort 3 was not initiated. With nivolumab alone, four patients achieved <ypT2ypN0 (26%), with two pathologic complete responses (pCR; 13%). With ipilimumab/nivolumab, three achieved <ypT2ypN0 (20%), with one pCR (7%). One patient after nivolumab and two after ipilimumab/nivolumab had durable clinical complete responses (CR) without cystectomy. Twelve-month recurrence-free survival (RFS) was 79% with nivolumab [95% confidence interval (CI), 61-100] and 61% with ipilimumab/nivolumab (95% CI, 39-95). Sequencing analyses suggest that NCOR1 alterations may be associated with improved clinical outcomes. Gene expression profiling indicated a potential association between tumor-infiltrating immune cells and longer RFS (log-rank P = 0.18); this was also observed in PURE-01 (P = 0.022). CONCLUSIONS:Among cisplatin-ineligible patients with MIBC, nivolumab alone was well tolerated. Ipilimumab/nivolumab caused toxicity that delayed cystectomy. Cases of progression before cystectomy indicated insufficient efficacy of pure neoadjuvant immunotherapy for unselected patients. Despite low response rates, some patients experienced sustained clinical CR without cystectomy.

TPS290 Background: Landmark randomized trials established the survival benefit of adding D to ADT in mCSPC. Building on this foundation, subsequent trials (PEACE-1 and ARASENS) demonstrated further survival improvements when androgen receptor pathway inhibitors (ARPI) were added to the ADT + D backbone. However, the independent contribution of D within the triplet regimen remains unclear given lack of prospective evaluation. Additionally, tumor suppressor gene (TSG) alterations involving TP53, PTEN, and RB1 are frequently observed in PC and are associated with aggressive biology and poor outcomes, providing an opportunity to enhance prognostic accuracy and optimize selection of candidates for treatment intensification. We hypothesize that adding D to ADT plus A will improve survival compared to ADT + A in mCSPC and stratification by TSG status may identify different treatment effects across molecular subgroups. Methods: ASPIRE is an open-label, two-arm, randomized phase 3 study to assess the benefit of D added to ADT plus A for patients with mCSPC. Eligible patients include those with confirmed adenocarcinoma of the prostate with evidence of distant metastatic disease on conventional imaging and are appropriate to receive D. Patients with metachronous low-volume disease or PSMA-PET only disease are excluded. Candidates must have next-generation sequencing (NGS) results from a CLIA-certified tissue test available for TSG status stratification at registration. Eligible patients will receive ADT plus A (240 mg PO daily) +/- D for up to 6 cycles until progression of disease by PCWG3/RECIST version 1.1 criteria. Prior therapy with ADT (with or without ARPI) initiated within 120 days before registration is permitted. The primary endpoint is overall survival (OS). Secondary endpoints include OS by TSG status; radiographic progression-free survival (rPFS); time to castration resistance; symptomatic skeletal event–free survival; time to worsening of disease-related symptoms (NCCN-FACT FPSI-17); safety and tolerability (CTCAE v5.0); PSA90 response at 6 weeks and 6 months; time to PSA progression; and objective response rate in patients with measurable disease. The study will enroll 1200 patients and is currently actively accruing patients. Clinical trial information: NCT06931340 .

e16569 Background: Diet can modulate the gut microbiome and may affect ICB efficacy. In a cohort of pts with mUC on ICB, we previously reported associations of longer PFS with high dietary fiber and low dietary fructose. These associations were independent of clinical factors. However, observational studies can be affected by confounding, so we sought to determine if confounding by predictors of metabolic health explained our prior findings. Methods: In a retrospective analysis of a prospectively collected cohort, we leveraged dietary data collected with the Harvard Willett Food Frequency Questionnaire from pts with mUC initiating ICB at Memorial Sloan Kettering to assess for associations between PFS and baseline BMI, daily caloric intake (cal/d), and dietary glucose. These were treated as continuous variables and log transformed if skewed. Associations with PFS were assessed by univariate (UV) & multivariable (MV) Cox proportional hazards regression. Results: From 2/2021-6/2022, 38 pts eligible for analysis enrolled. Median follow-up was 10.4 months with 27 PFS events. Median (with interquartile range) for each variable of interest: fiber 17 g/day (13-22); fructose 15 g/day (12-31); BMI 26.5 kg/m2 (23.4-30.4); glucose 15 g/day (11-25); cal/d 1,597 (1,091-1,939). Visceral metastases were present in 21 (55%) pts, and 5 (13%) had prior ICB. BMI, glucose, and cal/d were not associated with PFS in UV models (BMI HR 0.27, 95% CI 0.02-3.29, p=0.31; glucose HR 1.46, 95% CI 0.74-2.86, p=0.27; cal/d HR 0.74, 95% CI 0.30-1.79, p=0.50), models adjusted for tumor mutational burden, Bellmunt risk factors, and prior ICB (BMI HR 0.34, 95% CI 0.02-4.96; p=0.43; glucose HR 1.3, 95% CI 0.63-2.64, p=0.49; cal/d HR 0.51, 95% CI 0.19-1.40, p=0.19), nor models adjusted for fiber and fructose intake (BMI HR 0.27, 95% CI 0.02-3.11; p=0.29; glucose HR 0.51, 95% CI 0.06-4.61, p=0.55; cal/d HR 0.67, 95% CI 0.13-3.45, p=0.63). Associations of fiber and fructose intake with PFS persisted after adjusting for BMI, glucose, and cal/d (Table 1). Conclusions: In mUC, BMI, cal/d, and dietary glucose were not associated with PFS on ICB. Confounding by these variables did not account for associations of high dietary fiber and low dietary fructose with longer PFS. Hazard ratios (HR) with 95% confidence intervals (CI) for PFS. All MV models included fiber & fructose. Dietary variable UV models Model for fiber & fructose together Adjusted for BMI Adjusted for glucose intake Adjusted for cal/d Fiber, g/day 0.98 (0.94-1.02); p = 0.28 0.90 (0.84-0.97); p = 0.005 0.91 (0.85-0.97); p = 0.004 0.91 (0.84-0.97); p = 0.008 0.91 (0.84-0.996); p = 0.04 Fructose, log(g/day) 1.66 (0.89-3.09); p = 0.11 5.02 (2.06-12.23); p = 0.0004 5.06 (2.08-12.32); p = 0.0004 8.65 (1.16-64.30); p = 0.035 5.44 (2.08-14.18); p = 0.0005

CD40 agonism activates dendritic cells (DCs) and promotes T-cell antitumor immunity, but how tumor immune contexture shapes clinical responses remains unclear. We conducted a phase I study of intravesical Fc-optimized CD40 agonist 2141-V11 in 25 patients with bacillus Calmette-Guérin (BCG)-unresponsive non-muscle invasive bladder cancer (NMIBC), integrating spatial immune profiling. 2141-V11 was well tolerated, with no dose-limiting toxicities, and the recommended phase 2 dose was 70 mg. Clinical activity was observed, with 40% responses seen at any timepoint and durable benefit (median 20 months). Baseline tumors were immune-inflamed and enriched in tertiary lymphoid structures (TLS), whose abundance and maturity were not associated with response. Instead, response correlated with TLS composition, with enrichment of DC–CXCL13+ CD4+ and CD8+ T cell triads in responders, whereas non-responders showed increased DC-regulatory T cell proximity. These findings identify spatially organized DC–T cell interactions as a determinant of response to CD40 agonism in BCG-unresponsive NMIBC.

Bladder cancer brain metastasis (BrM) remains a poorly characterized clinical entity in neuro-oncology. The current study combines clinicopathologic and genomic sequencing data to identify prognostic factors in bladder cancer BrM. BrM patients were identified from a single-center retrospective cohort study at an NCI-designated Comprehensive Cancer Center consisting of 1,847 patients who were diagnosed with bladder cancer and received a brain magnetic resonance imaging between January 1, 2000 and December 31, 2024. The study cohort did not consist of an unselected population of all patients with bladder cancer. Genomic data was obtained from the Memorial Sloan Kettering-Integrating Mutation Profiling of Actionable Cancer Targets clinical sequencing platform. A multivariable Cox proportional hazards regression model was constructed by including baseline covariates that were associated with overall survival from univariable analysis at p < 0.05. 133 patients (median age at BrM diagnosis = 69.5 years [range 34.8–92.7], 99 [74.4

5018 Background: mCRPC is characterized by marked molecular heterogeneity and variable clinical outcomes. Circulating tumor DNA (ctDNA) profiling provides a noninvasive approach to capture tumor genomic alterations with potential prognostic value. We developed and validated a ctDNA-based prognostic model for rPFS using data from the Alliance A031201 (NCT01949337) trial, distinct from our published CG model of overall survival (Halabi et al. Eur Urol 2025). Methods: We analyzed ctDNA from 776 pts enrolled in the A031201 trial that randomized men with chemotherapy-naïve mCRPC to enzalutamide +/- abiraterone acetate and prednisone. Baseline cell free DNA samples from 776 pts were sequenced using the AR-ctDETECT assay. The primary endpoint of this analysis was rPFS. Proportional hazards model was used to assess the association of each genetic alterations and clinical variables with rPFS. Random survival forest (RSF) incorporating CG and clinical (C) variables only were trained and evaluated using 10-fold cross-validation. Model performance was assessed using integrated time-dependent area under the ROC curve (itAUC) and net reclassification improvement (NRI). Results: Higher ctDNA aneuploidy fraction, ctDNA positivity, and multiple pathogenic genomic alterations were associated with worse rPFS. Among prevalent alterations, AR enhancer gain and AR gain were strongly prognostic, with median rPFS of 13.7 vs 27.3 months (mos) and 13.5 vs 27.0 mos for pts with and without alterations, respectively. The rPFS prognostic model included gains in AR, AR enhancer, MYC, CCND1, and FOXA1 , and losses in PTEN, TP53, and LRP1B based on RSF . PTEN loss, gains in AR enhancer, MYC, along with hemoglobin, PSA and alkaline phosphatase showed the largest contribution to risk prediction. Mean itAUC for C model was 0.66 (95% confidence interval [CI] 0.62-0.70), mean itAUC for CG model was 0.73 (95% CI: 0.69–0.77). At 22 mos (median rPFS), NRI comparing CG model with C model was 0.30 (95% CI: 0.19-0.36). Pts were stratified into low-, intermediate-, and poor-risk groups and showed markedly distinct rPFS outcomes, with median rPFS of 39.2, 25.2, and 12.8 mos, respectively. The hazard ratios for low vs poor risk was 0.25 (95% CI: 0.20–0.31) and for intermediate vs poor risk 0.46 (95% CI: 0.38-0.55). Conclusions: A ctDNA-based prognostic model integrating CG features robustly stratifies rPFS risk in pts with mCRPC treated with first line AR pathway inhibitor therapy. This approach may support individualized risk assessment and inform trial design around treatment intensification combinations and therapeutic decision-making. External validation in independent cohorts is warranted. Support: U10CA180821, U10CA180882; R01CA256157; R01CA174777; https://acknowledgments.alliancefound.org. Clinical trial information: NCT01949337 .

4578 Background: EV, alone (mono) or with pembrolizumab (EVP), is an established therapy for metastatic urothelial carcinoma (mUC). However, the activity with EV rechallenge after prior exposure and subsequent disease progression is not well defined. Methods: A retrospective institutional database of patients (pts) treated with EV mono (n=395) or EVP (n=256) for mUC was reviewed to identify pts who received EV rechallenge after disease progression. EV-free interval (EVFI) was defined as the time from last EV1 (initial) dose to first EV2 (rechallenge) dose. Investigator-assessed objective response rate (ORR; CR/PR), progression free survival (PFS), overall survival (OS), and duration of response (DoR, from first documented response) were summarized descriptively. Results: Of 42 pts identified, EV1 regimen was mono for 62% (n=26; EV1-mono) and EVP for 38% (n=16; EVP1). Median age was 77, 76% were male, and 45% had upper tract primary. Most pts (n=35; 83%) discontinued EV1 for toxicity, predominantly peripheral neuropathy. Median follow up from EV2 initiation was 26 mo (95% CI 17-NR). At rechallenge, 10 pts (24%) switched EV regimens (EV1-mono to EVP2, n=5; EVP1 to EV2-mono, n=5). EV2 was initiated at 1.25 mg/kg in four pts (9.5%), 1.0 mg/kg in 13 pts (31%), and ≤0.75 mg/kg in 25 pts (60%). Among response-evaluable pts (n=39), ORR to EV2 was 21% (8/39; 95% CI 9%-36%), including 2 CRs (5%), both with prior CR to EV1. No responses were observed in pts with SD/PD to EV1 (0/8; 0%), or those with EV1 PFS <6 mo (0/11; 0%). Among pts with long (>12 mo) or intermediate (6-12 mo) EVFI, ORR to EV2 was 33% (4/12) and 31% (4/13), respectively. No responses were reported with short (<6 mo) EVFI (0/14; 0%). EV2 was initiated at 0.75 mg/kg in 5/8 pts with CR/PR. Median PFS and OS estimated from EV2 start were 2.8 and 18.5 mo. Among pts with CR/PR, median DoR was 7.0 mo (95% CI 3.5-NR). At last follow up, three pts remained on EV2; 39 pts discontinued EV2 due to progression (74%, n=29), toxicity (23%, n=9) or pt preference (2.6%, n=1). Conclusions: In a real-world cohort, EV rechallenge demonstrated modest activity, primarily in pts with prior benefit from EV and EVFI >6 mo. These observations underscore the need for further studies to clarify the effects of repeated EV exposure and optimize patient selection. This is particularly relevant given efforts to mitigate toxicity and the emerging issue of recurrent disease after perioperative EVP. AllN = 42 EV1-monoN = 26 EVP1N = 16 EV1 ORR (95% CI) 79% (63%–90%) 85% (65%–96%) 69% (41%–89%) Median PFS, mo (95% CI) 8.3 (7.5–9.9) 8.6 (7.7–19.1) 7.6 (5.5–11.9) Median Time on EV, mo [IQR] 4.9 [3.7–7.2] 5.1 [4.1–8.7] 4.1 [2.4–5.7] EV2 ORR (95% CI)NE 8/39; 21% (9%-36%)3 5/25; 20% (7%–41%)1 3/14; 21% (5%–51%)2 Median PFS, mo (95% CI) 2.8 (2.6–5.1) 2.7 (2.6–5.3) 4.1 (2.6–NR) Median OS, mo (95% CI) 18.5 (11.6–29.7) 18.5 (11.0–29.7) 15.2 (12.6–NR) Median Time on EV, mo [IQR] 2.8 [1.9–5.6] 2.5 [1.9–5.1] 3.4 [1.9–6.1]

e16589 Background: Treatment with enfortumab vedotin and pembrolizumab (EV-P) is highly effective in patients (pts) with advanced urothelial carcinoma. The optimal local therapy for residual or oligoprogressive disease in the bladder after EV-P remains to be defined. Radiation (RT) to the bladder is highly effective in appropriately selected pts with muscle-invasive bladder cancer. Here, we report outcomes for pts receiving definitive bladder or pelvic RT after EV-P. Methods: A retrospective institutional database of pts treated with EV-P (n = 256) was reviewed to identify pts who received bladder or pelvic RT. RT-intent (consolidative, oligoprogressive, palliative) was coded. Investigator-assessed local control (LC), defined by recurrence at a RT-treated site, progression free survival (PFS), and overall survival (OS) were estimated from RT start. Treatment-related toxicity was retrospectively graded using CTCAE both acutely (within 3m of RT) and at 6-month intervals. Results: 36 pts received EV-P and RT to the bladder/pelvis. 24 were treated with definitive intent (for oligoprogression on/after EV-P or to consolidate non-growing residual disease). 7 were excluded from further analysis for RT given before EV-P, after intervening therapy, or > 1 year after EV-P. For the remaining 17 pts, 10 were treated for oligoprogression and 7 to consolidate residual disease. At diagnosis, 5 had M1b disease, 7 were M1a, 4 were N+M0, and 1 had N0M0 MIBC. Median follow-up was 11.7m. The median total duration of EV-P treatment was 4.1 months. All pts held EV during RT. RT was delivered to the bladder (n = 12, 4 with nodal coverage) or pelvic LNs alone (n = 5 patients); 7/17 received concurrent chemotherapy (all gemcitabine). After RT, 7/17 continued EV and/or P. LC at 12 and 24 months was 75%. 4/17 (24%) had local failure in the RT field at any time. After RT, median LC, PFS, and OS were 32.1 (95% CI NR-NR), 8.1 (95%CI 3.9-NR) and not reached (NR) (95%CI 11.9-NR) months. Median LC, PFS, and OS were not met for the consolidation patients and were 32.1, 3.9 and 11.9 months for oligoprogression pts. There were no Grade 3+ GU and 1 grade 3+ GI toxicities (a colo-vesical fistula in the context of disease progression with colonic invasion). Conclusions: Definitive bladder-pelvic RT after EV-P was well-tolerated with promising disease control, particularly in those patients receiving RT for residual non-progressing disease. Clinical outcomes and follow-up summary. Cohort Local Control Progression-Free Overall Survival All Patients 32.1 [NR to NR] 8.1 [3.9 to NR] NR [11.9 to NR] Consolidative NR [NR to NR] NR [NR to NR] NR [NR to NR] Oligoprogression 32.1 [NR to NR] 3.9 [2.2 to NR] 11.9 [8.4 to NR] NR: Not Reached; Values are Median [95% Confidence Interval] in months.