Radiation-induced change of PD-1/PD-L1 immune checkpoint in mouse and human colorectal cancer models

Abstract

Nadir, representing minimal tumor volume

and Regrowth, with regrown tumors after RT. Defining the Day 1 as an initiation of RT, tumor tissues were obtained on Day 1, 6, 12, and 22, and Day 1, 6, 10, and 20, with single ablative and fractionated dose regimen, respectively. PD-L1 expression on tumor cells, PD-1 expression on tumor-infiltrating CD4+ and CD8+ T cells, and proportions of tumor-infiltrating CD4+ and CD8+ T cell populations were estimated using flow cytometry analysis. Considering human data, we conducted paired analysis using pre-CRT biopsies and the corresponding post-CRT resected tissues of 123 rectal cancer patients undergoing preoperative CRT followed by surgery between 2005 and 2012. Immunohistochemistry of PD-L1, PD-1, and CD8 was analyzed along with other clinicopathologic features and survival outcomes. Results: PD-L1 expression on mouse tumor cells surged within a few days after completion of RT, followed by abrupt decreases on the Nadir and Regrowth phases (P < .001 and equal to .002 for single ablative and fractionated RT, respectively). PD-1-positivity (%) on CD4+ T cells was not significantly different according to different time points (P = .656 and .223 for single ablative and fractionated RT, respectively). On the contrary, PD-1-positive proportions (%) in CD8+ T cells sharply increased, and the high-level was sustained until the Regrowth phase (P < .001 for all paired comparisons between Pre-RT and others). During RT response of Early and Nadir time points, CD4+ T cells decreased, but CD8+ T cells increased. The alterations were reversed at Regrowth phase, with increasing and decreasing again in CD4+ and CD8+ T cell populations, respectively (P < .001 all paired comparisons between Pre-RT and others). In the immunohistochemistry of rectal cancer, PD-L1 expression levels and density of CD8+ tumor-infiltrating lymphocytes (TILs) increased after CRT (P < .001 for both). Considering PD-1 expression, its pre-CRT intensity was scanty, but markedly increased after CRT. With cutoffs using each median value, sustained higher expression of PD-L1 at pre- and post-CRT (high-to-high) was associated with less increase in the density of CD8+ TILs (P = .020). Patients with sustained high-to-high PD-L1 expression had poorer overall survival (OS) and disease-free interval (DFI) in univariate Kaplan-Meier analysis (P = .018 and .029, respectively), with inferior DFI in low-to-low density CD8+ TILs (P = .010). In multivariate analysis, two subgroups with high baseline PD-L1 expression level showed worse OS, but the highest risk was observed with the high-to-high alteration (hazard ratio [HR] 8.34, 95% confidence interval [CI] 1.85–37.53 and HR 11.03, 95% CI 2.33–52.29 for high-to-low and high-to-high, respectively). Conclusions: This study verified radiation-induced immunologic shift toward increases of the PD-1/PD-L1 checkpoint activity and density of CD8+ TILs. However, the change was maximal at the early phase of RT response, which highlights the need of concurrent combinatory strategy of PD-L1 blockade and RT. The alteration profiles of checkpoint-related molecules identified the subset of patients with poor prognosis, suggesting potential candidates who can benefit from combining checkpoint inhibitors.

Introduction: Recent progress in immunotherapy has introduced programmed death-1 (PD-1)/ programmed death-ligand 1 (PD-L1) blockade as a novel target to eradicate tumors, but its use has been mainly confined to recurrent or metastatic settings. Radiotherapy (RT), a major part of anti-cancer treatment, directly kills tumor cells, and subsequent anti-tumor immune responses are up-regulated. However, immunologic impacts of RT on PD-1/PD-L1 immune checkpoint activity has not been much investigated. This study evaluated RT-induced alterations of the PD-1/PD-L1 checkpoint molecules based on a murine colon carcinoma and human rectal cancer treated with preoperative chemoradiotherapy (CRT). Methods: CT26 colon carcinoma cell line was subcutaneously inoculated on the right hind leg of BALB/c mice. Based on tumor growth curves after irradiation of 15 Gy x 1 fx or 5 Gy x 3 fx, mouse tumors were surgically resected on 4 different time points: Pre-RT, non-irradiated status just prior to initiation of RT

Early, the early phase of RT response