1465 Multiplex imaging identifies unique immunophenotypic and spatial characteristics associated with response to immune checkpoint inhibitors (ICIs) in metastatic urothelial cancer (mUC)

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)

References

Bellmunt J, de Wit R, Vaughn DJ, Fradet Y, Lee J-L, Fong L, et al. Pembrolizumab as Second-Line Therapy for Advanced Urothelial Carcinoma. New England Journal of Medicine. 2017;376:1015–26. Galsky MD, Arija JÁA, Bamias A, Davis ID, Santis MD, Kikuchi E, et al. Atezolizumab with or without chemotherapy in metastatic urothelial cancer (IMvigor130): a multicentre, randomised, placebo-controlled phase 3 trial. The Lancet. 2020;395:1547–57. Akturk G, Sweeney R, Remark R, Merad M, Gnjatic S. Multiplexed Immunohistochemical Consecutive Staining on Single Slide (MICSSS): Multiplexed Chromogenic IHC Assay for High-Dimensional Tissue Analysis. Methods Mol Biol. 2020;2055:497–519. Sharma P, Retz M, Siefker-Radtke A, Baron A, Necchi A, Bedke J, et al. Nivolumab in metastatic urothelial carcinoma after platinum therapy (CheckMate 275): a multicentre, single-arm, phase 2 trial. The Lancet Oncology. 2017;18:312–22. Maier B, Leader AM, Chen ST, Tung N, Chang C, LeBerichel J, et al. A conserved dendritic-cell regulatory program limits antitumour immunity. Nature. 2020;580:257–62. Bankhead P, Loughrey MB, Fernández JA, Dombrowski Y, McArt DG, Dunne PD, et al. QuPath: Open source software for digital pathology image analysis. Sci Rep. 2017;7:16878. Braun DA, Hou Y, Bakouny Z, Ficial M, Sant’ Angelo M, Forman J, et al. Interplay of somatic alterations and immune infiltration modulates response to PD-1 blockade in advanced clear cell renal cell carcinoma. Nat Med. 2020;26:909–18.

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).