Predictive biomarkers for immune checkpoint inhibitors (ICIs) are largely identified retrospectively, but their prospective clinical utility remains unproven. Here, we report DUTRENEO, a prospective randomized phase 2 trial testing whether a retrospectively validated 18-gene bulk tumor inflammation signature (TIS) can guide neoadjuvant ICI therapy in muscle-invasive bladder cancer. The trial does not meet its primary endpoint, and this demonstrates that bulk gene-expression stratification does not sufficiently enrich for responders. To define the biology underlying this failure, we generate single-cell spatial transcriptomic profiles of 377 genes in ∼5.4 million cells across large tissue areas. Response is governed by spatial architectures invisible to bulk assays, including CD8+ T cell proximity to cancer cells, localized checkpoint co-expression within epithelial cancer-rich neighborhoods, and fibroblast-rich immune-excluded communities in non-responders. We provide a quantitative framework showing that ≥77 genes and ≥3-mm tissue diameter regions preserve predictive spatial signal at scalable throughput. The registration details of the trial are EudraCT 2017-002246-68.
Cell reports. Medicine. 2026 Jun 23 [Epub ahead of print]
Enrique Grande, Mustafa Sibai, Daniela Grases, Elena Andrada, Oscar Reig, Marc Escobosa, Ainara Azueta, Daniel Castellano, Javier Puente, Jaime Martínez de Villarreal, Albert Font, Teresa Alonso-Gordoa, Raquel Benítez, Ane Moreno-Oya, Mario Álvarez-Maestro, Javier Burgos, M Angel Climent, Mario Domínguez, Patricia Galván, Isabel Galante, Juan F García, Elena Perez, Xavier García Del Muro, Félix Guerrero-Ramos, Miriam Marqués, Pablo Maroto, Jesús M Paramio, Alvaro Pinto, Aleix Prat, Núria Malats, Ignacio Durán, Eduard Porta-Pardo, Francisco X Real
Medical Oncology Department, MD Anderson Cancer Center Madrid, Facultad de Medicina, Universidad Francisco de Vitoria, Madrid, Spain. Electronic address: ., Josep Carreras Leukaemia Research Institute, Badalona, Spain; Barcelona Supercomputing Center, Barcelona, Spain., Josep Carreras Leukaemia Research Institute, Badalona, Spain., Epithelial Carcinogenesis Group, Spanish National Cancer Research Centre-CNIO, Madrid, Spain; Centro de Investigación Biomédica en Red Cáncer, Madrid, Spain., Medical Oncology Department, Hospital Clinic, Barcelona, Spain; Translational Genomics and Targeted Therapies in Solid Tumours, IDIBAPS, Barcelona, Spain; Department of Medicine, University of Barcelona, Barcelona, Spain., Pathology Department, Hospital Universitario Basurto, Bilbao, Spain; Translational Prostate Cancer Research Lab, Biobizkaia Health Research Institute, Barakaldo, Spain., Servicio de Oncología Médica, Hospital 12 de Octubre, Madrid, Spain., Medical Oncology Department, Hospital Clínico San Carlos, Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, Spain., Medical Oncology Department, Institut Català d'Oncologia, Badalona Applied Research Group in Oncology (B-ARGO Group), Institut Investigació Germans Trias i Pujol (IGTP), Badalona, Spain., Medical Oncology Department, Hospital Universitario Ramón y Cajal, Madrid, Spain., Centro de Investigación Biomédica en Red Cáncer, Madrid, Spain; Genetic and Molecular Epidemiology Group, Spanish National Cancer Centre-CNIO, Madrid, Spain., Hospital La Paz, Madrid, Spain., Urology Department, Hospital Ramón y Cajal, Alcalá University, IRYCIS, Madrid, Spain., Servicio de Oncología Médica, Fundación Instituto Valenciano de Oncología, Valencia, Spain., Servicio de Urología, Hospital Marqués de Valdecilla, Santander, Spain., Translational Genomics and Targeted Therapies in Solid Tumours, IDIBAPS, Barcelona, Spain; Cancer Institute and Blood Disorders, Hospital Clinic, Barcelona, Spain., Servicio de Urología, Hospital Clínico San Carlos, Madrid, Spain., Pathology Department, MD Anderson Cancer Center, Madrid, Spain., Institut Català d'Oncologia. IDIBELL, University of Barcelona, Barcelona, Spain., Urology Department, Hospital 12 de Octubre, Madrid, Spain., Oncologia Médica, Hospital Sant Pau, Barcelona, Spain., Molecular and Translational Oncology, CIEMAT, Biomedical Research Institute, University Hospital "12 de Octubre", CIBERONC, Madrid, Spain., Medical Oncology Service, Hospital Universitario La Paz - IdiPAZ, Madrid, Spain., Translational Genomics and Targeted Therapies in Solid Tumours, IDIBAPS, Barcelona, Spain; Department of Medicine, University of Barcelona, Barcelona, Spain; Cancer Institute and Blood Disorders, Hospital Clinic, Barcelona, Spain., Hospital Universitario Marqués de Valdecilla, IDIVAL, Santander, Spain., Josep Carreras Leukaemia Research Institute, Badalona, Spain; Barcelona Supercomputing Center, Barcelona, Spain. Electronic address: ., Epithelial Carcinogenesis Group, Spanish National Cancer Research Centre-CNIO, Madrid, Spain; Centro de Investigación Biomédica en Red Cáncer, Madrid, Spain; Departament de Medicina i Ciències de la Vida, Universitat Pompeu Fabra, Barcelona, Spain. Electronic address: .