Abstract 4532: Spatial profiling delineates tumor-T-cell dynamics in anti-PD-1-refractory melanoma treated with ipilimumab or ipilimumab plus nivolumab
Authors: Deen Nataly Naser Al , Campbell Katie M. , Medina Egmidio , Gonzalez Cynthia R. , Scumpia Philip O. , Ribas Antoni 
Published: April 2023

Abstract
Background: Immune checkpoint blockade (ICB) therapies promote anti-tumor activity by facilitating CD8 T-cell activation at the tumor site (e.g., anti-PD-1), or in the lymph node, promoting T-cells trafficking to the tumor site (e.g., anti-CTLA-4). Focusing on patients with metastatic melanoma refractory to anti-PD-1 therapy who received ipilimumab ± nivolumab (ipi±nivo), we hypothesize that combination therapy could overcome anti-PD-1 primary resistance in some patients, by facilitating T-cell trafficking and infiltration into the tumor.
Methods: Visium spatial transcriptomics using 18,000 RNA plex (n=10), multiplexed ion beam imaging (MIBI) using 32 protein plex (n=4), and CosMx single cell imaging using 1000 RNA plex (n=2) were performed on paired baseline (n=5) and on-therapy biopsy samples (n=5). Paired samples were derived from patients with complete response to ipi+nivo (n=3), partial response to ipi+nivo (n=1), and no response to ipi monotherapy (n=1).
Results: Visium spatial transcriptomics revealed a decrease in melanoma gene expression clusters and an increase in expression of immune clusters. Further characterization of the immune clusters revealed signatures of CD4+ and CD8+ T cells displaying stronger activation markers, in particular cytotoxic markers and interferon gamma clusters, along with M1 macrophage transcripts. However, further analysis of the paired biopsies in the ipi monotherapy non-responder revealed a concomitant increase in the immune-suppressive M2 macrophages, tolerogenic DCs, regulatory T cells, and exhausted T cells, which was not observed in the ipi+nivo responders. Confirmatory studies performed in two patients (ipi+nivo responder vs ipi non-responder) using MIBI confirmed a decrease in the tumor burden, and large increases in CD8 (199X in ipi+nivo vs 1.52X in ipi), CD4 (22.3X in ipi+nivo vs 8.56X in ipi), and T cells (26.1X in ipi+nivo vs 2.13X in ipi). Focusing on the non-responder (ipi) paired biopsies, CosMx revealed heterogeneous tumor clusters, with little to no immune infiltration in the baseline biopsy, and a decrease in tumor burden with increased markers of immune response and immune suppression in the on-therapy biopsy.
Conclusion: Integrated single cell spatial analyses, on the RNA and protein levels, revealed increased immune cell recruitment to the tumor site in responders to anti-CTLA-4 + anti-PD-1 therapy compared to a non-responder to anti-CTLA-4 monotherapy, and may provide clues to cancer-immune cell interactions in response to ICB. The presence of immune suppressive clusters at the tumor site in the on-therapy biopsy in the anti-CTLA-4 monotherapy would support our hypothesis that while anti-CTLA-4 facilitates T-cell trafficking and infiltration into the tumor site, anti-CTLA-4 alone without anti-PD-1 may not be sufficient to overcome anti-PD-1 primary resistance in some patients.
Citation Format: Nataly Naser Al Deen, Katie M. Campbell, Egmidio Medina, Cynthia R. Gonzalez, Philip O. Scumpia, Antoni Ribas. Spatial profiling delineates tumor-T-cell dynamics in anti-PD-1-refractory melanoma treated with ipilimumab or ipilimumab plus nivolumab. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4532.