Abstract 109: Multi-modal single-cell analysis of immunotherapy-experienced cutaneous and mucosal melanoma
Authors: Biermann Jana , Wang Yiping , Fan Linyue , Ho Patricia , Amin Amit D. , Melms Johannes C. , Tagore Somnath , Hoffer-Hawlik Kevin , Shah Parin , Shaikh Neha , Ramaradj Priyanka , Zhang Mingxuan , Ribas Antoni , Robert Caroline , Azizi Elham , Izar Benjamin 
Published: June 2023

Abstract
Immune checkpoint blockade (ICB) targeting CTLA-4 or the PD1/PD-L1 axis have become a standard of care therapy for most patients with metastatic melanoma. However, for patients with resistance to front-line ICB, especially those with BRAFWT tumors who received combination therapy, subsequent therapeutic options remain limited. Thus, understanding how ICB alters the tumor ecosystem, and thereby may create new vulnerabilities, is key to understanding effective therapies for ICB-experienced patients. Here, enabled through innovations in biospecimen research, we performed multi-modal single-cell RNA, T-cell receptor and single-cell spatial transcriptomics, coupled with whole-genome sequencing (WGS) of 42 melanoma patient biopsies. This cohort includes 7 patients with paired before- and on-/post-treatment biopsies, and spanned treatment-naïve metastatic cutaneous melanoma patients (n=10), on-ICB (aPD-1, n=7; aCTLA-4, n=1; combination therapy, n=2; total, n=10), and post-ICB (aPD-1, n=10; aCTLA-4, n=5; combination therapy, n=6; L19IL2+L16TNF, n=1; total, n=22). This cohort further includes 6 rare samples of mucosal melanoma (oral, n=4; rectal, n=2). In total, we profiled &gt;320,000 transcriptomes, including &gt;240,000 malignant and ~80,000 non-malignant cells, and 14,714 matched TCRs. We used a series of novel analytical approaches, including KINOMO to mitigate biases introduced by differences in gene dosage, and novel artificial intelligence frameworks, to discover differentially expressed genes and gene programs associated with treatment exposures and treatment sites. Compared to untreated specimens, there is an increase in antigen presentation (Wilcoxon rank-sum test, P=0) and reduction in lineage expression (Wilcoxon rank-sum test, P=0) in ICB-experienced tumors. Furthermore, mucosal melanomas harbor unique gene regulatory networks characterized by expression of immune evasive programs (e.g. SOX4; MAST, P=0) and exhibit lower expression of oxidative phosphorylation (Wilcoxon rank-sum test, P=0). Together, these data indicate a high degree of cellular adaption and plasticity. Through integration of single-cell, spatial and WGS, we further identify cancer clones defined by their aneuploidy patterns that confer geographically restricted immune evasion with a distinctly cold tumor microenvironment (TME), despite global T cell activation and clonal expansion. This suggest significant intralesional heterogeneity with respect to response to ICB, which may be explained by cancer cell-intrinsic factors rather than insufficient T cell responses to ICB. This work provides initial insights into spatio-temporal changes induced by ICB in cancer cells and the TME in a relatively large patient cohort. Our results indicate that functional and spatial variability exists in these tumors and may provide avenues for novel therapeutic development in ICB-experienced patients.
Citation Format: Jana Biermann, Yiping Wang, Linyue Fan, Patricia Ho, Amit D. Amin, Johannes C. Melms, Somnath Tagore, Kevin Hoffer-Hawlik, Parin Shah, Neha Shaikh, Priyanka Ramaradj, Mingxuan Zhang, Antoni Ribas, Caroline Robert, Elham Azizi, Benjamin Izar. Multi-modal single-cell analysis of immunotherapy-experienced cutaneous and mucosal melanoma [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 109.