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Multiplexed FRET-FLIM Profiling of Immune Checkpoint Interactions Predicts Response to Atezolizumab in Urothelial Carcinoma

Camacho, L.; Cacho-Navas, C.; Agüero, J.; Batmunkh, B.; Gracia, J. M.; O Sullivan, K.; Rementeria, M.; Miles, J.; Gumuzio, J.; Aguirre, F.; Martin Algarra, S.; de Andrea, C. E.; Parker, P. J.; Calleja, V.

2026-09-03 oncology
10.64898/2026.09.01.26361904 medRxiv
Show abstract

Immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have shown great promise in treating bladder cancer and are now part of the standard treatment for advanced disease. However, many patients still fail to respond to treatment and at present many biomarkers are assessed but have yet shown only limited results. Therefore, with the advent of combination treatments and the increase of immune related adverse event, the search for reliable predictive biomarkers is paramount. Using a multiplexed enhanced FRET-FLIM based technique (QF-Pro) we quantified the interaction of PD-1/PD-L1, CTLA-4/CD80 and TIGIT/CD155 immune checkpoints in a pre-treatment TMA of 46 patients treated with atezolizumab. The association between higher PD-1/PD-L1 ICP interaction state and treatment efficacy was demonstrated in the male sample cohort, where it identified patients with better PFS. Conversely, patients exhibiting higher CTLA-4/CD80 engagement had a worse response to atezolizumab. Remarkably, the dual assessment of patients with high PD-1/PD-L1 and low CTLA-4/CD80 allowed to identify the best responders. These results indicate that the monitoring of patients immune profile in urothelial carcinoma might be critical in identifying patients who may benefit from combination therapy.

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