Exploration of tumor microenvironment using multiplexed immunohistochemistry and its association with response in advanced or metastatic renal cell carcinoma treated with immune checkpoint inhibitors

Abstract

Immune checkpoint inhibitors (ICIs) such as anti-programmed-death-1 (PD-1) and cytotoxic T lymphocyte protein-4 (CTLA4) inhibitors, are important treatment options for patients with advanced renal cell carcinoma (RCC). The tumor microenvironment (TME) in ICI-treated patients remains largely unknown. In this study, 71 patients were treated with ICIs between July 2015 and June 2020: 20 patients were treated with nivolumab plus ipilimumab as first-line therapy and 51 patients were treated with nivolumab monotherapy as second/third-line therapy. Multiplexed immunohistochemistry (mIHC) was performed to define the TME, which included various T cell subsets, B cells, macrophages, and dendritic cells. In the nivolumab plus ipilimumab group, the objective response rate (ORR) and disease control rate (DCR) were 47.1% and 64.7%, respectively. The density of CD8+ cytotoxic T cells (P=0.027), specifically CD103+ CD8+ tissue-resident T cells (P=0.046), CD137+ CD8+ T cells (P=0.005), Foxp3- CD4+ helper T cells (P=0.025), Foxp3+ CD4+ regulatory T cells (P=0.014), and CD68+ CD206- M1 macrophages (P=0.007) were significantly higher in responders than in non-responders. Patients who experienced initial disease progression had a lower infiltration of CD68+ CD206- M1 macrophages than those who did not (P=0.008). The median progression-free survival (PFS) was 11 months, and a high density of CD8+ cytotoxic T cells (P=0.030), Foxp3- CD4+ helper T cells (P=0.036), CD137+ CD8+ cytotoxic T cells (P=0.024), and CD68+ CD206- M1 macrophages (P=0.054) was associated with better PFS. In the nivolumab group, ORR and DCR were 38.8% and 61.2%, respectively, and median PFS was 6.3 months. Response to nivolumab monotherapy was not significantly associated with the infiltration of T cell subsets and B cells, but patients who had initial disease progression with nivolumab had a higher infiltration of CD68+ CD206+ M2 macrophages than those who did not (P=0.012). A high density of CD137+ CD8+ cytotoxic T cells (P=0.016), CD137+ CD4+ cytotoxic T cells (P=0.088), and a low density of CD68+ CD206+ M2 macrophages (P=0.067) were associated with better PFS. When quantifying the infiltration of immune cells according to the International Metastatic RCC Database (IMDC) risk groups, CD68+ CD206+ M2 macrophages were more highly infiltrated in high-risk patients than in intermediate- or favorable-risk patients. The density of T cell subsets significantly correlated with that of CD68+ CD206- M1 macrophages and CD20+ B cells, and the density of PD-L1+ cells significantly correlated with that of various immune cells. Therefore, through comprehensive TME analysis, CD137-expressing T cell subsets and M1/M2 macrophages were significantly associated with ICIs efficacy. This suggests that CD137 could be a predictive marker for selecting treatment options, and novel strategies targeting CD137 or macrophages should be further explored in advanced RCC.