Abstract 4621: Distinct spatial and molecular maps in the tumour microenvironment of resistant melanoma patients treated with checkpoint inhibitor immunotherapy

Abstract Background: Checkpoint-based immunotherapies can achieve durable clinical responses in a variety of advanced cancers, including melanoma. However, clinical responses are not universal and there is still a substantial gap in understanding the mechanistic insights of immunotherapy resistance. This study aims to better understand patients’ resistance to immune checkpoint therapies by dissecting and tracing individual tumour and immune cell clones. Methods: Melanoma tumour dissociates and lesion matched formalin-fixed paraffin-embedded tissue samples were collected from patients with advanced melanoma receiving ipilimumab in combination with nivolumab; 2 patients were long-term responders, 2 had innate resistance and 1 acquired resistance. The molecular characteristics and spatial cellular relations associated with tumour and immune subsets in response to treatment were identified using single-cell indexing of transcriptomes and epitopes by sequencing and CODEX (CO-Detection by indEXing) multiplexed tissue imaging. Integration of spatial and molecular information was performed by combining reference-based phenotyping, Louvain clustering, and a mutual nearest neighbours-based transfer learning algorithm. Results: Pre-treatment melanoma biopsies from 5 patients (3 responding and 2 non-responding) along with a subsequent acquired resistance biopsy from an initially responding patient were analysed. Comparisons of cellular phenotypes identified higher proportions of gamma delta T cells (1.98% versus 0.2%) and enriched memory T cell profiles (CD3E, CD44, CD45RO, CD8) in responding compared to non-responding pre-treatment biopsies. Fibroblast from non-responding lesions overexpressed stromal related cytokine, IL1A (log2 fold change (FC) = 3.3) and CXCL1 (log2 FC = 1.4), which were enrichment at the tumour margin compared to responding lesions. Comparisons of a patient’s pre-treatment biopsy that initially responded to a subsequent progressing biopsy with acquired resistance had altered expression of transcript factor, ZFX (log2 FC = -1.59; adjusted p = 0.0119), and MBD2 (log2 FC = -1.5; adjusted p = 0.0225) within the patient’s memory T-cells. Conclusion: The combination of high dimensional single cell transcriptomics and spatial tissue imaging are effective tools to identify specific changes in cell phenotypes, intracellular signalling, and spatial location. This analysis identified reduced memory T cells, increased fibroblast, and altered cellular signalling were associated with resistance to immunotherapies. Citation Format: Camelia Quek, Aditya Pratapa, Ines Pires da Silva, Ghamdan Al-Eryani, Aaron Mayer, Nenad Bartonicek, Kate Harvey, Oliver Braubach, Robyn P.M. Saw, Jonathan Stretch, Kerwin F. Shannon, Alexander M. Menzies, Richard A. Scolyer, Georgina V. Long, Alexander Swarbrick, James S. Wilmott. Distinct spatial and molecular maps in the tumour microenvironment of resistant melanoma patients treated with checkpoint inhibitor immunotherapy. [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 4621.