Abstract 1026: A multiplex IHC evaluation of multiple immune checkpoint receptors and mismatch repair proteins in colorectal carcinoma
Authors: Yang George , Figueroa Sara , Tacha David , Douglas Cristin 
Published: August 2018

Abstract
Introduction: The recent success of checkpoint inhibitor therapies in clinical oncology practice has generated overwhelming enthusiasm for immunotherapeutics. Recent studies have suggested that patients with mismatch repair deficient (dMMR)/microsatellite instability-high (MSI-H) colorectal carcinoma (CRC) may benefit from anti-PD-1/PD-L1 inhibitors. In this study, we have evaluated a large panel of immune checkpoint receptors (ICRs) on CRC by multiplex immunohistochemistry (IHC). Design: Tissue sections were initially reviewed by H&E to confirm the presence of adequate pathological features including histology, grade and abundance of tumor-infiltrating lymphocytes (TILs). An MMR protein panel (MLH1, MSH2, MSH6, PMS2) was tested by IHC to determine dMMR/MSI-H status through loss of MLH1/PMS2 or MSH2/MSH6. Multiple ICRs, including CD4, CD8, CD137, CTLA-4, FOXP3, GITR, IDO1, LAG-3, PD-1, and PD-L1, were then tested by multiplex IHC on 18 CRC cases consisting of MSI-H CRCs (n=8) and microsatellite stable (MSS) CRCs (n=10) using four chromogenic end-points. Expression of ICRs in TILs was determined at four sites: tumor, invasive margin, peri-tumor, and tertiary lymphoid structures (TLS). ICR expression levels were determined by counting and averaging positive TILs in three high-power fields (400X) and categorized in the following manner: 0 (negative, no expression), 1 (weak, 1-25 cells), 2 (moderate, 26-50 cells), 3 (high, &gt;50 cells). Results: Five cases of MSS (50%) and two cases of MSI-H showed 10-30% PD-L1 positive tumor cell expression. The remaining cases were negative for PD-L1. All cases of MSI-H and MSS demonstrated high expression of CD8 in tumor, invasive margin, peri-tumor and TLS. Five MSI-H cases showed moderate to high PD-1, and three MSI-H cases were weak for PD-1. All MSS cases showed weak to moderate PD-1 expression. CD4, FOXP3, CTLA-4, IDO1, LAG-3, and CD137 were weak to moderate in all MSI-H and MSS cases. Within the TLS, the predominant cellular components were CD8+, CD4+ and PD-1+ T cells in all cases. Conclusion: In the present study, a greater number of MSS CRCs expressed PD-L1 than MSI-H CRCs. PD-1 was positive in all CRC cases with variable expression between MSI-H and MSS CRCs. There appears to be no significant difference in TIL expression of additional immune checkpoint receptors tested in MSI-H colorectal carcinomas and MSS colorectal carcinomas. Multiplex technology appears to allow for superior evaluation of the synergistic relationship between immune checkpoint receptors as well as the interconnection between inhibitory barriers to CD8+ effector T cells and regulatory T cells developed by immune checkpoint receptors within each of the tumoral regions. Further study of expression patterns of immune checkpoint receptors may provide useful information for clinical management of patients with colorectal carcinoma.
Citation Format: George Yang, Sara Figueroa, David Tacha, Cristin Douglas. A multiplex IHC evaluation of multiple immune checkpoint receptors and mismatch repair proteins in colorectal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1026.