Abstract 2533: In-depth multi-omic profiling of immune cells in cancer patients developing immune-related adverse events
Authors: Shek Dmitrii , Gao Bo , Lai Joey , Gloss Brian , Nagrial Adnan , da Silva Ines Pires , Carlino Matteo S. , Read Scott A. , Ahlenstiel Golo 
Published: March 2024

Abstract
Background: The clinical efficacy of immune-checkpoint inhibitors is complicated by the risk for immune-related adverse events (irAEs) that can involve any organ systems. To date, (1) we cannot predict irAE risk reliably at patient level and (2) irAEs remain a diagnosis of exclusion as there are no specific clinical or biological markers. The goal of this study was to elucidate intricate transcriptomic and proteomic signatures of immune cells in patients evolving irAEs.
Methods: This pilot trial was conducted as part of a prospective multicenter cohort study (NCT04631731). The study cohort consisted of n=71 patients who were treated with ICIs across Blacktown and Westmead Hospitals, NSW, Australia. Peripheral blood mononuclear cells (PBMCs) were collected from both pre-treatment and after 6-8 weeks post-ICI commencement. Samples were further utilized for RNA sequencing, flow cytometry and Chromium 3’ gene expression analysis (10x Genomics).
Results: The median age was 67 years old with the 78% (n=56) of Caucasian origin. The predominant number of patients were treated with dual ICIs. N=21 patients experienced irAEs. Firstly, a differential analysis established a set of genes which were significantly (FDR&lt;0.01) dysregulated between patients with (group A) and without irAEs (group B) and were mostly related to cell cycle and regulation. Notably, phospholipase D signaling pathway was exclusively upregulated in group A and is known to be linked to B cell expansion. Secondly, a cell enrichment analysis within the group A revealed a significant increase of CD4+ and CD8+ T central memory (Tcm) cells upon the toxicity onset as compared to baseline stage. Thirdly, the flow cytometry data established that group A had (1) higher expression of CD27 on CD4+ T cells, a member of Traf-linked tumor necrosis factor receptor family essential for T cells’ maturation and (2) lower expression of inhibitory receptor LAG3 (CD223) which is crucial for maintaining homeostasis of immune system through suppression of T cell proliferation. Finally, we sequenced CD45+ cells from n=4 patients from group A and n=4 from group B at both pre- and post-treatment stages. The unsupervised clustering established 17 different clusters in our single cell dataset with cluster 11 was unique only to patients from group A. The differential analysis established that a set of n=62 genes was significantly upregulated (p&lt;0.05) in cluster 11. Functional annotation established that B cell activation and proliferation pathways were enriched by those genes further stressing B cells’ role in irAEs development.
Conclusions: Our real-world cohort study established significant molecular and cellular differences between patients with and without irAEs. Further research in larger cohorts is imperative to validate the established findings and to elucidate the precise mechanisms contributing to the development of irAEs.
Citation Format: Dmitrii Shek, Bo Gao, Joey Lai, Brian Gloss, Adnan Nagrial, Ines Pires da Silva, Matteo S. Carlino, Scott A. Read, Golo Ahlenstiel. In-depth multi-omic profiling of immune cells in cancer patients developing immune-related adverse events [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2533.