PD05.08. T-Cell Inactivation in Esophageal Adenocarcinoma Is Mediated by ISG15 Related CD3 Loss

Abstract

Esophageal Cancer: Molecular Biology/Pathology Esophageal adenocarcinoma (EAC) remains a deadly cancer and is often characterized by treatment resistance. Previous work by our group has demonstrated that loss of T-cells is correlated to worse survival. Our study aims to investigate the role of Interferon-stimulated gene 15 (ISG15) in loss of T-cell functionality within the EAC tumor microenvironment (TME). Treatment naïve esophageal tumor samples were prospectively collected from patients undergoing resection for EAC and a tissue microarray was created utilizing Barrett’s esophagus (BE), low-grade (LGD), high-grade (HGD) and tumor specimens. Multiplex fluorescent immunohistochemistry (mfIHC) was performed to identify epithelial tumor cells (cytokeratin positive) and immune cells in the tumor microenvironment (effector T-cell, regulatory T-cell, macrophages). A spatial G-function, which is a nearest-neighbor distribution function, was then used to quantify the likelihood of tumor cells encountering anergic T-cells. We also analyzed a cohort of treatment resistant patient from a TMA generated from 115 patients who had residual tumor after neoadjuvant therapy. Visium spatial transciptomic analysis of these tissues were used to measure ISG15 levels. Lastly, we performed western blot analysis on T-cell lines undergoing ISG15 knockdown to determine the effects on CD3 expression. Samples were collected from 64 patients with BE, 50 with LGD, 54 with HGD, and 95 with EAC. mfIHC demonstrated a significant loss of CD3 (p<0.04) expression with transition from HGD to EAC. Higher CD3-Tumor engagement in EAC patients was also correlated to improved overall survival. ISG15 expression was found to be elevated in EAC compared to dysplastic tissue and the increase was seen when we evaluate ISG15 in a cohort of 113 treatment resistant patients. Lastly, western blot analysis demonstrated that knockdown of ISG15 resulted in increased CD3 expression, whereas ISG15 overexpression promotes CD3 (epislon, gamma, and delta chains) degradation. Esophageal cancer is shaped by an immune suppressive TME. This suppression is mediated by loss of CD3 within T-cells which drives anergy. We believe the main driver of T-cell loss in EAC is via ISG15. This has implication for prognosis and may impact response to neoadjuvant therapy. Further studies should be designed to target ISG15 mediated T-cell loss to improve survival in EAC.