Abstract 2345: Colorectal peritoneal metastases: Exploring the immune landscape and the potential of immunotherapy using organoid models
Authors: Vignesh Narasimhan, Toan Pham, Kasmira Wilson, Susan Woods, Daniel Worthley, Jeanne Tie, Michael Michael, Alexander Heriot, Robert Ramsay
Journal: Cancer Research (2019)

Abstract Background: Colorectal cancer (CRC) is the third most common cancer worldwide. Up to half the patients with CRC will develop metastatic disease, with an associated 5-year survival of 13.5%. The peritoneum is a common site for metastases, but carries the worst prognosis among all sites. Most patients with colorectal peritoneal metastases (CRPM) are inoperable, leaving systemic chemotherapy as the only treatment option. With systemic therapy alone, only 4% of patients with CRPM are alive at 5 years. Immunotherapy, while effective in a number of cancers, remains unexplored in peritoneal disease. Here, we aim to firstly explore the immune landscape of CRPM, and subsequently evaluate the role of immunotherapy in CRPM using organoid models. Methods: Fresh microsatellite stable CRPM tissue from 25 patients was used for flow cytometry and multi- spectral immunohistochemistry (OPAL) to assess the immune cell infiltrate and density in the tumour microenvironment. Remaining tissue was used to develop organoid models, and to culture and expand patient matched tumour-infiltrating lymphocytes (TILs). To evaluate the immune profile further, organoids were exposed to interferon gamma for 48 hours and stained for MHC-I and PD-L1. Organoids were co-cultured in a cytotoxic assay with TILs to assess the cytotoxic ability of the lymphocytes. Organoids were then co-cultured with TILs with the addition of an anti PD-1 antibody to assess the role of checkpoint blockade. To assess TIL specificity, organoids were co-cultured with TILs with MHC-I antibody, and FACS sorted populations of CD8+, CD4+, CD56+ and CD3+CD56+ cells. Results: FACS analysis revealed a heterogenous population of immune cells in CRPM with a significantly higher T cell infiltrate compared to a predominantly myeloid cell infiltrate in normal peritoneum (T cell 30% v 11%, p &amp;lt;0.05; myeloid cells 20% v 64%, p&amp;lt;0.05). There were high levels of T cell memory and activation markers such as CD45RO, CD69 and HLA-DR. PD-1 expression on T cells, whilst variable, was upto 75% in some patients, suggesting a possible role for anti PD-1 therapy in selected cases. OPAL demonstrated a significant immune infiltrate in the tumour microenvironment in many cases, with a strong stromal immune infiltrate. Co-culture of organoids and matched TILs demonstrated successful organoid killing by the TILs. In patients with high T cell PD-1 expression on FACS, addition of an anti PD-1 antibody revealed significantly increased cytotoxic killing of the organoids. Conclusion and future direction: There appears to be a significant immune infiltrate in many cases of CRPM that can be potentially harnessed with the use of checkpoint blockade. These highly promising results need to be expanded with a larger cohort Citation Format: Vignesh Narasimhan, Toan Pham, Kasmira Wilson, Susan Woods, Daniel Worthley, Jeanne Tie, Michael Michael, Alexander Heriot, Robert Ramsay. Colorectal peritoneal metastases: Exploring the immune landscape and the potential of immunotherapy using organoid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2345.