Integration of peripheral blood- and tissue-based biomarkers of response to immune checkpoint blockade in urothelial carcinoma

As predictive biomarkers of response to immune checkpoint inhibitors (ICIs) remain a major unmet clinical need in patients with urothelial carcinoma (UC), we sought to identify tissue-based immune biomarkers of clinical benefit to ICIs using multiplex immunofluorescence and to integrate these findings with previously identified peripheral blood biomarkers of response. Fifty-five pretreatment and 12 paired on-treatment UC specimens were identified from patients treated with nivolumab with or without ipilimumab. Whole tissue sections were stained with a 12-plex mIF panel, including CD8, PD-1/CD279, PD-L1/CD274, CD68, CD3, CD4, FoxP3, TCF1/7, Ki67, LAG-3, MHC-II/HLA-DR, and pancytokeratin+SOX10 to identify over three million cells. Immune tissue densities were compared to progression-free survival (PFS) and best overall response (BOR) by RECIST version 1.1. Correlation coefficients were calculated between tissue-based and circulating immune populations. The frequency of intratumoral CD3⁺LAG-3⁺ cells was higher in responders compared to nonresponders (p = 0.0001). LAG-3⁺ cellular aggregates were associated with response, including CD3⁺LAG-3⁺ in proximity to CD3⁺ (p = 0.01). Exploratory multivariate modeling showed an association between intratumoral CD3⁺LAG-3⁺ cells and improved PFS independent of prognostic clinical factors (log HR −7.0; 95% confidence interval [CI] −12.7 to −1.4), as well as established biomarkers predictive of ICI response (log HR −5.0; 95% CI −9.8 to −0.2). Intratumoral LAG-3⁺ immune cell populations warrant further study as a predictive biomarker of clinical benefit to ICIs. Differences in LAG-3⁺ lymphocyte populations across the intratumoral and peripheral compartments may provide complementary information that could inform the future development of multimodal composite biomarkers of ICI response. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.