Abstract A077: Sustained immune response to anti-PD-1 therapy for oral premalignant lesions (OPLs) in a carcinogen-induced oral cancer mouse model
Authors: Monteiro de Oliveira Novaes Jose Augusto , Hirz Taghreed , Pisegna Marlese A. , Hwu Patrick , Heymach John V. , William William N. 
Published: August 2018

Abstract
PD-L1 expression occurs in OPLs in humans and is associated with increased cancer risk (William et al., ASCO 2017). These data suggest a contribution of the PD-1/PD-L1 axis to immune evasion of OPLs, and warrant evaluation of anti-PD-1 immunotherapy for cancer prevention. We conducted this preclinical study in a carcinogen-induced mouse model of oral cancer to test the hypothesis that a short course of anti-PD-1 therapy, given at a time point when only OPLs are present (and before invasive cancer development), could lead to enhanced and sustained immune responses. We treated C57BL/6 mice with the carcinogen 4-nitroquinoline 1-oxide (4-NQO) for 8 weeks in drinking water. Eight weeks after 4-NQO discontinuation, a group of mice (N=6) was sacrificed for assessment of invasive and noninvasive tongue lesions. A second group of mice was treated with either anti-PD-1 antibody (N=10) or IgG (N=10) for a total of 3 doses every 3 days initiating 8 weeks after the cessation of 4-NQO. We sacrificed these animals 56 days after the last dose of treatment, harvested cervical lymph nodes, and prepared and stained cell suspensions with fluorochrome-labeled antibodies against CD45, CD3, CD8, Granzyme B (GZB), PD-1, TIM-3, Lag-3, ZombieNirt (live/dead discrimination) for flow cytometric evaluation. We also assessed serial H&E-stained cross-sections of the tongues harvested at that same time point for development of OPLs and cancers. Student’s t-test and Fisher's exact test were used for statistical comparisons between groups. At 8 weeks after 4NQO cessation, 6 out of 6 mice developed tongue hyperplasia or dysplasia. No invasive lesions were identified, indicating this to be an ideal time point to study/initiate treatment strategies addressing OPLs. Anti-PD-1 therapy led to a sustained increase in the proportion of CD8+|GZB+ cytotoxic T lymphocytes (CTLs) when compared to IgG (mean 26.9% ± 4.4% versus 14.4% ± 2.3%, respectively; p= 0.025) in cervical lymph nodes harvested 56 days after treatment discontinuation. We also observed an increase in the proportion of CD8+|PD-1+ CTLs (mean 22.3% ± 2.08% versus 13.3% ± 1.42%, respectively; p= 0.0026) and a trend towards an increase in CD8+|TIM-3+|LAG-3+ CTLs (mean 5.9% ± 1.5% versus 2.5% ± 0.7%, respectively; p= 0.0733). This immune profile is indicative of activated CTLs with limited/no signs of exhaustion. At this late sacrifice time point, frankly invasive oral cancers had developed in 70% vs 40% of IgG and anti-PD1-treated mice, respectively (p=0.36). Short-term anti-PD-1 immune checkpoint inhibitor therapy in the context of OPLs led to a sustained increase in the population of granzyme B+ CTLs in cervical lymph nodes, accompanied by a trend towards a reduction in the incidence of oral cancer. These results suggest that immune checkpoint-targeted therapy can reverse immune suppression already present at the preinvasive stage, and support the ongoing Immune Prevention of Oral Cancer (IPOC) clinical trial evaluating pembrolizumab in high-risk OPLs (NCT02882282). Additional preclinical studies are under way to assess if an immune response to anti-PD1 therapy can occur at earlier time points, and lead to pronounced prevention of oral cancers at a stage when the (irreversible) malignant transformation process induced by the potent 4-NQO carcinogen is not yet far advanced.
Citation Format: Jose Augusto Monteiro de Oliveira Novaes, Taghreed Hirz, Marlese A. Pisegna, Patrick Hwu, John V. Heymach, William N. William. Sustained immune response to anti-PD-1 therapy for oral premalignant lesions (OPLs) in a carcinogen-induced oral cancer mouse model [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A077.