Background ICIs increase survival in mUC1 but only a subset (~15–25%) of patients experience durable disease control.2 Differences in the tumor microenvironment (TME) might underlie such differential responses. However, the complex network of cellular interactions within the TME that associate with response and resistance to ICIs remains underexplored.
Methods Multiplex Immunohistochemical Consecutive Staining on a Single Slide (MICSSS)3 was performed on UC specimens (N=40) from CheckMate 2754 prior to treatment with nivolumab. 9 immunohistochemical stains (PD-L1, CD8, CD3, pan-cytokeratin, fibronectin, CD68, FAP, DC-LAMP,5 CD11b) were sequentially performed on a single slide per patient. Image processing, whole slide annotation (median 464,554 cells/slide), and intra- and extra-tumoral compartment training, were performed using QuPath (figure 1).6 Responders (CR, PR) and non-responders (SD, PD) were defined per RECIST v1.1. Immunophenotypic designations of ‘inflamed’, ‘excluded’, and ‘desert’ were defined via tumor margin CD8 analysis.7 Lymphoid aggregates were identified morphologically with dense CD3 positivity. Single-cell spatial analysis was performed defining neighborhoods as the 25 nearest neighboring cells.
Results TME characterization demonstrated inter-tumoral heterogeneity, both in the intra- and extra-tumoral compartments (figure 2). Responders contained >2-fold increased intra-tumoral CD8 cells, though no cell types were significantly altered in comparison to non-responders. In contrast, extra-tumoral CD3, CD8, CD3CD8-, DC-LAMP (PD-L1- and PD-L1+), and PD-L1+ CD11b cells were significantly enriched in responders (figure 3). Inflamed tumors were more prevalent and excluded/desert tumors less prevalent in responders, with inflamed tumors containing increased intra-tumoral T cell and DC-LAMP infiltration. There were no significant differences in infiltrate composition between inflamed responders and inflamed non-responders, while excluded/desert responders demonstrated enrichment for extra-tumoral DC-LAMP cells (PD-L1- and PD-L1+) and intra-tumoral PD-L1- DC-LAMP cells as compared to excluded/desert non-responders (figure 4). Responder tumors also contained an increased density of lymphoid aggregates, which were found in closer proximity to tumor regions, were associated with increased survival, and were comprised of a greater degree of DC-LAMP cells (figure 5). Spatial analysis of extra-tumoral cells identified a unique immune and tumor-enriched PD-L1+ neighborhood (cluster 0) predominant in responders, and a distinct CD11b and tumor-based neighborhood devoid of PD-L1 and other immune cells (cluster 2) predominant in non-responders (figure 6).
Conclusions Multiplex immunohistochemistry identified unique immunophenotypic and spatial TME features specific to mUC responders to ICI. Both increased infiltration and the geographic arrangement of T cells, dendritic cells, and PD-L1 positivity, particularly in the extra-tumoral compartment, may prove key in identifying responders to ICI.
Trial Registration This is a secondary translational analysis from NCT02387996 (CheckMate 275, CA209–275)
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Ethics Approval The initial clinical trial NCT02387996 obtained appropriate ethics approval, and was conducted in accordance with Good Clinical Practice guidelines defined by the International Conference on Harmonisation. All participants provided informed consent before taking part in the study based on the principles of the Declaration of Helsinki. Approval was granted from local institutional review boards or ethics committees at each center (as published).
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