Radical cystectomy is the most commonly used definitive local treatment for muscle-invasive bladder cancer (MIBC), yet it carries substantial perioperative complication risk, alongside major changes in urinary and sexual function. Chemoradiotherapy (CRT) is used as a bladder-sparing alternative but is usually reserved for small, solitary tumors. Highly active systemic induction therapy could enable bladder preservation for patients with more advanced tumors and reduce recurrence risk. We previously demonstrated high activity of preoperative ipilimumab (3 mg kg-1) plus nivolumab in patients with stage III MIBC. Given this high activity, the single-arm, multicenter phase 2 INDIBLADE trial aimed to provide effective bladder-sparing treatment to patients with stage II/III (cT2-4aN0-2, n = 50) MIBC, using induction ipilimumab (3 mg kg-1) plus nivolumab followed by CRT. After a median follow-up of 28.7 months, the primary endpoint of estimated 2-year bladder-intact event-free survival (BI-EFS) was met at 78% (95% confidence interval (CI): 0.67-0.9, P < 0.001). Secondary endpoints included overall survival, safety and the predictive value of circulating tumor DNA (ctDNA). Two-year overall survival was 96% (95% CI: 0.91-1). Grade 3-4 immune-related adverse events occurred in 24% of patients; grade 3-4 CRT-related adverse events occurred in 7% of patients. Absence of detectable ctDNA after induction immunotherapy was associated with BI-EFS (hazard ratio 8.3, 95% CI: 1.38-50.36, P = 0.02). In conclusion, our results show that induction combination immunotherapy followed by CRT is an effective bladder-sparing treatment in MIBC, and response can be monitored by ctDNA. ClinicalTrials.gov identifier: NCT05200988 .

New therapeutic strategies for patients with therapy-refractory metastatic urothelial carcinoma (mUC) are still needed. In the phase I-II ICRA trial, a multicenter, open-label, phase Ib-II clinical trial (NCT03871036), we evaluated whether paclitaxel plus tremelimumab, with or without durvalumab, could induce a tumor response in mUC following platinum chemotherapy and immune checkpoint inhibition (ICI). Patients were randomized between three arms: A (n = 20) paclitaxel plus T750mg; B (n = 12) paclitaxel plus T300mg plus D1500mg; C (n = 12) T750mg. The primary outcome, objective response rate, was met with 26% in arm A (88% CI = [14,36%]) versus 17% (88% CI = [3,42%]) in arm B and 8% (88% CI = [0.3,33%]) in arm C. Secondary outcomes included safety, duration of response and survival. Grade 3-4 treatment-related adverse events occurred in (A) 45%, (B) 75%, and (C) 25% of patients. Murine experiments showed attenuated tumor outgrowth for paclitaxel plus anti-CTLA-4 compared to monotherapy with either agent. Transcriptomic analyses showed upregulation of inflammation signatures in on-treatment tumor tissue biopsies. This study demonstrated encouraging antitumor activity in therapy-refractory mUC treated with paclitaxel plus high-dose anti-CTLA-4 and suggests immune induction may still be possible after failure to anti-PD-(L)1 therapy.

Survival of patients with metastatic castration-resistant prostate cancer (mCRPC) depends on the site of metastatic dissemination. Patients with mCRPC were prospectively included in the CPCT-02 metastatic site biopsy study. We evaluated whole genome sequencing (WGS) of 378 mCRPC metastases to understand the genetic traits that affect metastatic site distribution. Our findings revealed that RB1, PIK3CA, JAK1, RNF43, and TP53 mutations are the most frequent genetic determinants associated with site selectivity for metastatic outgrowth. Furthermore, we explored mutations in the non-coding genome and found that androgen receptor (AR) chromatin binding sites implicated in metastatic prostate cancer differ in mutation frequencies between metastatic sites, converging on pathways that impact DNA repair. Notably, liver and visceral metastases have a higher tumor mutational load (TML) than bone and lymph node metastases, independent of genetic traits associated with neuroendocrine differentiation. We found that TML is strongly associated with DNA mismatch repair (MMR)-deficiency features in these organs. Our results revealed gene mutations that are significantly associated with metastatic site selectivity and that frequencies of non-coding mutations at AR chromatin binding sites differ between metastatic sites. Immunotherapeutics are thus far unsuccessful in unselected mCRPC patients. We found a higher TML in liver and visceral metastases compared to bone and lymph node metastases. As immunotherapeutics response is associated with mutational burden, these findings may assist in selecting mCRPC patients for immunotherapy treatment based on organs affected by metastatic disease. NCT01855477.

Details about methods used for mIF, DNA- and RNA isolation and sequencing and single-cell RNA sequencing

Perturbation response models (PRMs) predict transcriptional responses to interventions such as gene knockout or drug treatments. While simple baseline models match PRMs in reconstructing post-treatment profiles, we show that drug response predictors trained on PRM-generated profiles significantly outperform those trained on baseline or pre-treatment profiles. Unlike simple baseline models, PRMs preserve variation in response-relevant genes, making them a highly valuable tool for predicting drug response.

Patient fractions per molecular subtype for response groups a. Fraction of patients per molecular subtype (n=5) according to the consensus classification. b. Fraction of patients per molecular subtype (n=4) according to the TCGA classification. There were no neuro-endocrine subtypes in our patient cohort. Responders (<=ypT1N0) are depicted in blue, non-responders (ypT2-4aNx or ypTxN1-3) are depicted in orange.

Tumor mutational burden (TMB) in responders vs non-responders for ipilimumab-high and ipilimumab-low. Baseline TMB (somatic non-synonymous TMB per Mb) in the ipilimumab-high cohorts (cohort 1 and 2A; left panel) and the ipilimumab-low cohort (cohort 2B; right panel) for responders (blue; <=ypT1N0) and non-responders (orange; ypT2-4aNx or ypTxN1-3).

Consort diagram of available samples per analysis for each arm at baseline and on-treatment. One patient in arm 2A and 3 patients in arm 2B did not undergo cystectomy.

Platform studies optimize molecular profiling offering matched therapies to selected patients (pts). Prior studies have reported the activity of antiPD1/PD-L1 in solid tumor pts with high TMB without considering the underlying genetic background. Basket of Baskets (NCT03767075), is a platform trial from Cancer Core Europe. Pts underwent molecular profiling (iPROFILER) and were assigned to one of the modules (iBASKET). In module 1 arms, IO-naïve pts received atezolizumab according to alterations in DNA repair genes or high/intermediate TMB (see table). Pts provided paired biopsies, serial plasma, and stool for correlative studies. overall response rate at 12 weeks (ORR). This module used a Bayesian design. Arm size varied from 5 to 20 pts to identify an ORR from 30-50%. Exploratory endpoints: (i) correlation of response and clinical benefit with alterations observed by tumor WES, RNA-Seq, and proteomics, (ii) correlation of response with serial cfDNA and plasma proteomics. Among 84 pts included in arms 1A-1F, 79 were evaluable for response. Higher ORR and best ORR were observed in arm 1B and 1D with high TMB (see table). Nine additional responses were observed after 12 weeks. Responses were observed in pts with colorectal (4), bladder (2), lung (2), breast, ovarian, gastric, adrenocortical, anal, salivary gland cancer, basal cell carcinoma and carcinoma of unknown primary (1 each). Preliminary data suggests that early changes in cfDNA and plasma proteomics correlate with clinical benefit. WES and RNA-Seq data are expected for the meeting presentation. Higher response rate was observed in arm 1B and 1D high TMB, including pts with rare tumors and without reimbursed indication. Initial evidence suggests a correlation of cfDNA and plasma proteomics dynamics with clinical benefit. Irene Brana, Christophe Massard, Frans Opdam, Richard D. Baird, Luigi De Petris, Richard Schlenk, Claudio Vernieri, David Tamborero, Shubha Anand, Etienne Rouleau, Adria Lopez, Nikos Demiris, Paolo Nuciforo, Elena Provenzano, Daniel Huebschmann, Katrin Pfütze, Maria Pernemalm, Daniel Vis, Ana Vivancos, Valtteri Wirta, Peter Horak, Omar Saavedra, Marloes van Dongen, Emily Barker, Susana Aguilar, Elena Chavarria, Xenia Villalobos, Marta Rotxes, Arantxa Romero, Francisco Martínez-Jiménez, Alena Gros, Anna Pedrola, Cristina Viaplana, Neeltje Steeghs, Elena Garralda, Cancer Core Europe - Basket of Baskets study team, Alejandro Piris, Karim Fizazi, Fabrice Barlesi, Karen Barker, Michiel Heijden, Wilbert Zwart, Filippo de Braud, Elias Arner, Janne Lehtiö, Michael Baumann, Stefan Fröhling, Josep Tabernero, Javier Carmona, Jordi Rodon. Cancer Core Europe - Basket of Baskets - first results from module 1: Atezolizumab in genomically selected populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr CT093.

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, paraffin-embedded tumor tissue was analyzed using PD-L1 IHC (n = 51) and whole-exome and transcriptome sequencing (both n = 53) and correlated with response. Baseline infiltration of CD8+ T cells (n = 51) and at cystectomy (n = 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 ipilimumab-high, 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.

Mutational analysis DDR genes. Fraction and type of alterations for various DNA damage response genes in responders (blue) and non-responders (orange). The different treatment arms are depicted in light green (arm 1), dark green (arm 2A) and lilac (arm 2B).

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. CLINICAL TRIAL REGISTRATION NUMBER:NCT03387761.

The poly(ADP-ribose) polymerase inhibitor (PARPi) class of drugs represents a remarkable advance in the treatment of patients with homologous recombination-deficient tumours, but resistance remains a challenge1-5. Although most research has focused on the downstream consequences of PARPi exposure to tackle resistance, the immediate effect of PARP inhibition on the chromatin environment and its contribution to PARPi toxicity remains elusive. Here we show that PARP inhibition induces histone release from the chromatin. This presents a vulnerability of PARPi-resistant cancer cells, which require histone homeostasis mechanisms to sustain elevated DNA replication rates and survival. Through functional genetic screens, we identified NASP as a key factor in maintaining the stability of evicted histones via its TPR motifs. Loss of NASP renders tumour cells hypersensitive to PARPi treatment in vitro and in vivo, impairs replication fork progression and elevates levels of replication-associated DNA damage. Moreover, NASP acts together with the INO80 complex and the chaperoning activity of PARP1 to ensure efficient histone turnover and prevent the accumulation of lethal DNA damage. Collectively, our work reports on histone eviction as an immediate cellular response to PARPi treatment and provides a promising avenue for targeting histone supply pathways to overcome PARPi resistance.

Representativeness of Study Participants

180 Background: Overcoming ARSI resistance is an unmet need for mCRPC patients. Known resistance mechanisms include genetic alterations augmenting androgen receptor (AR) function (e.g., amplifications of the AR gene and its enhancer and AR mutations) and treatment-emergent evolution from an AR-driven to an AR-indifferent phenotype such as small cell neuroendocrine prostate cancer. Other mechanisms remain unexplored. Methods: To phenotype ARSI-resistant prostate cancer, we compared the RNA-seq profiles of paired metastatic biopsies from 31 mCRPC patients obtained before the initiation of first-line abiraterone or enzalutamide, and upon radiographic progression. The patient samples were obtained from two independent cohorts, the Stand Up To Cancer/Prostate Cancer Foundation (SU2C/PCF) West Coast Prostate Cancer Dream Team (WCDT) and a multi-institutional cohort from the Netherlands sequenced through the Hartwig Medical Foundation. We first evaluated RNA-seq data to identify the most differentially expressed genes and then performed survival analysis in the retrospective WCDT cohort (n=115, including 54 ARSI-naïve and 61 ARSI-experienced tumors) to assess the association between gene expression and overall survival, defined as the time interval from biopsy to death from any cause. Results: In the analysis of paired RNA-seq data from 31 mCRPC patients, SSTR1 was identified as the most significantly altered gene; SSTR1 expression was consistently decreased in post-treatment, ARSI-resistant tumors (log2[fold change]=-2.5, p=1.08×10 -16 ). SSTR1 encodes somatostatin receptor 1, a G-protein coupled receptor that, when activated by the endogenous peptide hormone somatostatin or exogenous somatostatin analogues, mediates anti-proliferative, anti-migratory, and anti-secretory effects on target cells. Retrospective analysis in the 115 WCDT patients demonstrated that low SSTR1 expression was associated with worse overall survival ( p=0.047); ARSI treatment improved survival for patients with SSTR1-high (≥ median) tumors (HR=0.29, p=0.0002) more significantly than those with SSTR1-low (< median) tumors (HR=0.67, p=0.17), with an interaction ( SSTR1 expression * ARSI treatment) towards statistical significance ( p=0.0598). A differential survival benefit of ARSI therapy with respect to SSTR1 expression was observed only among the 61 ARSI-experienced patients. Conclusions: SSTR1 expression is a potential predictive biomarker for response to ARSI in mCRPC, with higher SSTR1 expression predicting better response. This observation could be explained by an anti-tumor effect of SSTR1 signaling. Further work is warranted to prospectively validate these findings and to investigate SSTR1 as an actionable drug target to overcome ARSI resistance.

BACKGROUND Androgen receptor signaling inhibitors (ARSIs) have improved outcomes for patients with metastatic castration-resistant prostate cancer (mCRPC), but their clinical benefit is limited by treatment resistance.METHODS To investigate the mechanisms of ARSI resistance, we analyzed the whole-genome (n = 45) and transcriptome (n = 31) sequencing data generated from paired metastatic biopsies obtained before initiation of first-line ARSI therapy for mCRPC and after radiographic disease progression. We investigated the effects of genetic and pharmacologic modulation of SSTR1 in 22Rv1 cells, a representative mCRPC cell line.RESULTS We confirmed the predominant role of tumor genetic alterations converging on augmenting androgen receptor (AR) signaling and the increased transcriptional heterogeneity and lineage plasticity during the emergence of ARSI resistance. We further identified amplifications involving a putative enhancer downstream of the AR and transcriptional downregulation of SSTR1, encoding somatostatin receptor 1, in ARSI-resistant tumors. We found that patients with SSTR1-low mCRPC tumors derived less benefit from subsequent ARSI therapy in a retrospective cohort. We showed that SSTR1 was antiproliferative in 22Rv1 cells and that the FDA-approved drug pasireotide suppressed 22Rv1 cell proliferation.CONCLUSION Our findings expand the knowledge of ARSI resistance and point out actionable next steps, exemplified by potentially targeting SSTR1, to improve patient outcomes.FUNDING National Cancer Institute (NCI), NIH; Prostate Cancer Foundation; Conquer Cancer, American Society of Clinical Oncology Foundation; UCSF Benioff Initiative for Prostate Cancer Research; Netherlands Cancer Institute.

Combining drugs can enhance their clinical efficacy, but the number of possible combinations and inter-tumor heterogeneity make identifying effective combinations challenging, while existing approaches often overlook clinically relevant activity. We screen one of the largest cell line panels (N = 757) with 51 clinically relevant combinations and identify responses at the level of individual cell lines and tissue populations. We establish three response classes to model cellular effects beyond monotherapy: synergy, Bliss additivity, and independent drug action (IDA). Synergy is rare (11% of responses) and frequently efficacious (>50% viability reduction), whereas Bliss and IDA are more frequent but less frequently efficacious. We introduce "efficacious combination benefit"(ECB) to describe high-efficacy responses classified as either synergy, Bliss, or IDA. We identify ECB biomarkers in vitro and show that ECB predicts response in patient-derived xenografts better than synergy alone. Our work here provides a valuable resource and framework for preclinical evaluation and the development of combination treatments.

PURPOSE:Platinum-based chemotherapy and immune checkpoint inhibitors are key components of systemic treatment for muscle-invasive and advanced urothelial cancer. The ideal integration of these two treatment modalities remains unclear as clinical trials have led to inconsistent results. Modulation of the tumor-immune microenvironment by chemotherapy is poorly characterized. We aimed to investigate this modulation, focusing on potential clinical implications for immune checkpoint inhibitor response. EXPERIMENTAL DESIGN:We assessed immune cell densities, spatial relations, and tumor/stromal components from 116 patients with urothelial bladder cancer (paired data for 95 patients) before and after platinum-based chemotherapy. RESULTS:Several published biomarkers for immunotherapy response changed upon chemotherapy treatment. The intratumoral CD8+ T-cell percentage increased after treatment and was associated with increased TNFα-via-NF-κB signaling. The percentage of PDL1+ immune cells was higher after chemotherapy. An increase in chemo-induced changes that potentially inhibit an antitumor immune response was also observed, including increased fibroblast-based TGFβ signaling and distances from immune cells to the nearest cancer cell. The latter two parameters correlated significantly in posttreatment samples, suggesting that TGFβ signaling in fibroblasts may play a role in spatially separating immune cells from cancer cells. We examined specific chemotherapy regimens and found that treatment with methotrexate, vinblastine, doxorubicin, and cisplatin was associated with an increase in the macrophage cell percentage. Gemcitabine-containing chemotherapy was associated with upregulation of fibroblast TGFβ signaling. CONCLUSIONS:The opposing effects of platinum-based chemotherapy on the immune cell composition and stromal context of the tumor-immune microenvironment may explain the inconsistent results of clinical trials investigating chemotherapy and immune checkpoint inhibitor combinations in bladder cancer.

Supplementary Table S1. Representativeness of Study Participants