Abstract 179: Spatial and multiomics analysis reveals immune interactions as key drivers of immunotherapy outcomes in melanoma patients with in-transit metastases

Abstract Background: In-transit metastasis (ITM) of melanoma occurs between the primary tumor and draining lymph nodes. ITM can be managed by by adjuvant immunotherapy after resection, or immunotherapy alone when unresectable. Nevertheless, nearly half of ITM patients are resistant, which constitutes a clinical challenge. Multiomic spatial profiling offers insights into features influencing immunotherapy response, but the tumor microenvironment (TME) and biological determinants of ITM remain poorly defined. This study aims to characterize molecular and spatial TME attributes in ITM patients to improve understanding of immunotherapy response and resistance. Methods: Fifty-four samples from ITM patients treated with immunotherapy were collected at pre-treatment (PRE) and progression (PROG). Patients with a complete or partial response in the advanced setting, or recurrence-free survival over 12 months in the adjuvant setting, were classified as "responsive," and others as "resistant". Single-cell sequencing and 40-marker imaging were conducted on 18 matched tumor dissociates and FFPE samples at PRE (n=3 resistant, n=5 responsive) and PROG (n=10). Whole genome sequencing on 12 fresh frozen samples (n=4 resistant, n=8 responsive at PRE) were used to identify somatic variants. Results: While regional immune cell proportions were broadly similar across response groups, PRE responders showed increased intra- and peri-tumoral T cells, and surprisingly, reduced CD141+ dendritic cells (cDC1s) compared to resistant patients (P=0.024). K-means clustering identified 7 spatially resolved cellular neighborhoods (CNs) across all ITMs with heterogeneous cellular constitution; nevertheless, lymphocyte-enriched CNs were more dispersed in PRE responders and interacted with other immune and tumor CNs, while remaining peripheral in PRE resistant tumors. Despite having a lower DC density, pairwise interaction analysis highlighted significant interactions between cDC1s and CD14+ macrophages in PRE responders, and between macrophages and IDO1+ CD4+ T cells or Granzyme B+ CD8+ T cells (P<0.05, log2 fold change>0.2), suggesting enhanced macrophage-driven local T cell stimulation in the absence of DCs. Single-cell transcriptomics analysis comfirmed the reduced proportion of cDC1s in PRE responders compared to resistant ITM (P=0.006). Genomic profiling revealed EREG mutations exclusively in resistant ITMs, which has been linked to immunoregulationand tumor invasion through the EGFR pathway. Ongoing analysis explores interactions among DCs, macrophages, T cells, B cells, melanocytes, and endothelial cells to elucidate TME dynamics associated with immunotherapy outcomes. Conclusion: These findings deepen our understanding of tumor-immune interactions in ITM patients and inform personalized immunotherapy approaches tailored to their unique TME. Citation Format: Xinyu Bai, Camelia Quek, Ghamdan Al-Eryani, Sonny Ramkomuth, Louise Baldwin, Jiabao Tian, Nurudeen A. Adegoke, Ismael A. Vergara, Felicity Newell, Kate Harvey, John Reeves, Andrew J. Spillane, Nicola Waddell, Serigne N. Lo, Alexander M. Menzies, Ines P. da Silva, Umaimainthan Palendira, Georgina V. Long, Richard A. Scolyer, Alexander Swarbrick, James S. Wilmott. Spatial and multiomics analysis reveals immune interactions as key drivers of immunotherapy outcomes in melanoma patients with in-transit metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 179.