Immune profiling of advanced, recurrent metastatic endometrial cancer using high-dimensional time-of-flight mass cytometry (CyTOF)

Abstract

<h3>Objectives:</h3> The efficacy of treatments for advanced, recurrent metastatic endometrial cancers (EC) remains limited. In-depth, high-dimensional immune profiling of recurrent EC subtypes is much needed in order to describe the immune composition within the tumor microenvironment. <h3>Methods:</h3> A 2:1 randomized phase 2 trial was conducted comparing the combination of cabozantinib and nivolumab (Arm A) versus nivolumab (Arm B) in women with recurrent measurable EC (NCT03367741). A third exploratory cohort (Arm C) included EC patients with the carcinosarcoma subtype, and patients who had prior immunotherapy (IO). Fresh baseline core biopsies were collected and processed into single-cell suspensions for high-dimensional CyTOF analysis with a 36-marker immune profiling panel. Immune composition was determined by unsupervised single-cell clustering using PhenoGraph and visualized by uniform manifold approximation and projection (UMAP). The differential abundance (DA) of unique immune cell subsets present in the baseline biopsies was determined using the diffcyt-DA-EdgeR method. <h3>Results:</h3> Cabozantinib plus nivolumab demonstrates improved PFS and ORR compared to nivolumab in heavily pre-treated women with recurrent EC. Unsupervised clustering of CD45+ cells from baseline biopsies (n=40) using PhenoGraph resulted in 35 unique immune cell subsets defined their expression of lineage and activation/checkpoint markers (e.g. PD-1, PD-L1, TIGIT, 4-1BB, CD39, CD69 and others). Broadly, these PhenoGraph-defined subsets constitute the major immune populations including CD4 and CD8 T cells, regulatory T cells, γδ T cells, B cells, innate lymphoid/natural killer (NK) cells, and monocyte/macrophages and dendritic cells. In-depth CyTOF analysis of the tumor microenvironment identified a higher proportion of activated tissue-resident γδ T cells in patients who had prior IO and who benefit from the combination therapy (non-progressors, n=4; progressors, n=5; 6 log fold-change, adjusted <i>P</i>=0.001). We did not observe any statistically significant differences in the baseline immune composition between IO-naïve (Arm A and B) endometrioid (n=13), serous (n=7) and carcinosarcoma tumors (n=4). However, in comparison to endometrioid and serous tumors, carcinosarcoma tumors may trend towards a lower abundance (not significant) of CD45RA+CD27+CD28+ CD4 and CD8 T cells, and CD45RA+CD69- NK cells. All three histological subtypes had a similar abundance of activated, tissue-resident PD-1+TIGIT+CD69+CD103+ CD8 T cells. Microsatellite instability high (MSI-H; n=2; both endometrioid) tumors were excluded from our analysis. <h3>Conclusions:</h3> In comparison to nivolumab monotherapy, combination cabozantinib and nivolumab treatment improved PFS and ORR in recurrent EC patients. To our knowledge, this is the first study reporting in-depth immune profiling analysis of core biopsies from EC patients. The tumor immune microenvironment of EC is comprised of several immune populations with unique phenotypes related to their expression of activation and checkpoint markers.