Abstract 2143: Profiling exhausted T cells using Vectra® Polaris™multiplex immunofluorescence assay in HNSCC
Authors: Pollan Sara , Hanifi Arezoo , Nagy Mate , Stavrou Nicholas , Parnell Erinn , Gozo Maricel , Attanasio Nickolas , William Josette , Au Qingyan 
Published: September 2020

Abstract
Background: Head and neck squamous cell carcinoma (HNSCC) is a cancer with the ability to modulate the immune system to evade detection. It is the sixth most frequently diagnosed cancer with 550,000 new cases and 300,000 lives lost worldwide per year. New treatments for HNSCC are urgently needed as patients continue to experience a high mortality rate and low response to surgery and chemotherapeutic treatments. Part of the reason why HNSCC is difficult to treat is it upregulates the expression of immune-checkpoint signaling molecule TIGIT (T cell immunoreceptor with Ig and ITIM domains) to inhibit T cell activation in vivo. Emerging evidence shows TIGIT overexpression in the CD8+ and CD4+ T cells that infiltrate the tumor cells of HNSCC patients. TIGIT expression is also associated with up-regulation of immune-checkpoint ligands PD-1 (programmed cell death protein 1) and LAG-3 (lymphocyte-activation gene 3 aka CD223), markers of T-cell exhaustion. Altogether, activation of the TIGIT/PD-1/LAG-3 axis correlates with an immunosuppressive microenvironment as well as cancer development and progression. Although there is ample evidence that the upregulation of TIGIT decreases the immune response in HNSCC, only limited studies have been published that address the location, expression and co-expression of TIGIT, LAG-3 and PD-1 in the HNSCC microenvironment.
Methods: In this study, we sought to establish a robust report of immune cells in the tissue of patients with HNSCC. Using Vectra Polaris multiplex immunofluorescence (IF) assays, we studied T-cell exhaustion and T-cell expression in HNSCC patient tissue using a total of 9 markers essential in cancer immunology. Sequential tissue sections were stained in two panels of 6, an exhausted T cell panel comprised of TIGIT, PD-1, LAG-3, panCK, CD4 and CD8 and a T cell panel including CD3, FOXP3, CD45RO, panCK, CD4 and CD8.
Results: Multiplexing IF staining revealed a HNSCC histologic landscape characteristic of immune suppression in this study. The data demonstrated abundant T cells with TIGIT overexpression in the tissue microenvironment of HNSCC samples. Using Indica Halo algorithms, we quantified exhausted T cells (TIGIT+PD1+LAG3+CD4+CD3+, TIGIT+PD1+LAG3+CD8+CD3+), T helper cells (CD3+CD4+), T cytotoxic cells (CD3+CD8+), T regulatory cells (CD3+CD4+FoxP3), memory T-cells (CD3+CD4+CD45RO) and anergic T-cells (PD1+CD8+) within the tumor and the stromal regions.
Conclusion: Currently AB154, a fully humanized immunoglobulin G1 monoclonal antibody targeting human TIGIT is in phase I clinical trials in HNSCC patients and BGB-A1217, an anti-TIGIT monoclonal antibody in combination with anti-PD-1 monoclonal antibody Tislelizumab is in a Phase 1/1b clinical trial in patients with advanced solid tumors. The Vectra Polaris imaging reported in this study identifies T cell composition in the tumor microenvironment of patients facing high mortality.
Citation Format: Sara Pollan, Arezoo Hanifi, Mate Nagy, Nicholas Stavrou, Erinn Parnell, Maricel Gozo, Nickolas Attanasio, Josette William, Qingyan Au. Profiling exhausted T cells using Vectra® Polaris™multiplex immunofluorescence assay in HNSCC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2143.