Spatial relationships in the urothelial and head and neck tumor microenvironment predict response to combination immune checkpoint inhibitors

Immune checkpoint inhibitors (ICI) currently achieve remarkable clinical results in urothelial cancer (UC). However, the relationship between the tumor microenvironment (TME), usually characterized by immune cell density, and response to ICI is unclear. We quantified the TME immune cell densities and spatial relationships (SRs) using the multiplex immunofluorescence data of 24 UC pre-treatment tumor resections. We described SRs by approximating the 1-NN distance distribution with a Weibull distribution and evaluated the association between TME metrics (spatial and density parameters) and ipilimumab+nivolumab response. Immune cell density did not discriminate between response groups. However, the Weibull SR metrics of CD8+ T-cells or macrophages to their closest cancer cell were positively associated with response. CD8+ T-cells close to B-cells were characteristic of non-response. The G- function, a threshold dependent alternative SR metric, yielded variable effect sizes and statistical power in association studies with response. We validated our SR response associations in a cohort of head and neck tumors with a comparable treatment design. Our data confirm that SRs, in contrast to density metrics, are strong biomarkers of response to ICIs, a finding with significant translational relevance. ### Competing Interest Statement MvdH received research funding from Bristol Myers Squibb, AstraZeneca, 4SC and Roche, and consultancy fees from Bristol Myers Squibb, Roche, Merck Sharp " Dohme, Merck, AstraZeneca, Pfizer, Janssen and Seattle Genetics which were all paid to the Netherlands Cancer Institute. LFAW received research funding from Genmab, which was all paid to the Netherlands Cancer Institute. CLZ received research financial support from Bristol Myers Squibb to fund the IMCISION trial, which was all paid to the Netherlands Cancer Institute. The rest of the authors have no conflicts of interest related to this work.