Background: The immune checkpoint PD-1 and its ligand PD-L1 are involved in the induction of immunological tolerance of solid tumors including oral squamous cell carcinoma (OSCC). The aim of the study was to establish the clinical and prognostic significance of PD-L1 in OSCC. Methods: Tissue microarrays of 125 resected OSCC were stained with two different commercially available PD-L1 antibodies (clones E1L3N and 22C3), alongside PD-1 immunostaining. Results: PD-L1 expression in more than 10% of tumor cells was associated with poorer survival, and established as a clinically relevant cut-off point. This relevant PD-L1 expression was detected in 10% to 15% OSCC specimens depending on the anti-PD-L1 antibody, and showed an inverse correlation with tobacco and alcohol consumption. We consistently found that PD-L1 expression was associated with tumor recurrence and lower disease-specific survival. Multivariate analysis further revealed that neck node metastasis (HR 2.304; P = 0.009) and tumor PD-L1 expression (HR 2.571; P = 0.01) were significant independent factors for poor prognosis. Conclusions: PD-L1 expression in more than 10% of tumor cells was a significant and independent factor of poor prognosis in OSCC. Impact: PD-L1 expression in more than 10% of tumor cells was consistently established as a clinically relevant cut-off point by using two different antibodies. Remarkably, PD-L1 expression emerges as an independent poor prognosis marker in patients with OSCC.

Tumor-associated macrophages (TAMs) can be polarized into antitumoral M1 and protumoral and immunosuppressive M2 macrophages. This study investigated the clinical relevance of TAM infiltration in oral squamous cell carcinoma (OSCC), evaluating CD68 (M1 and M2 macrophage marker) and CD163 expression (M2 macrophage marker) in the tumor nests and surrounding stroma. Immunohistochemical analysis of both stromal/tumoral CD68⁺ and CD163⁺ TAMs was performed in paraffin-embedded tissue specimens from 125 OSCC patients, and correlated with clinical data. Potential relationships with the expression of cancer stem cell (CSC) markers and PD-L1 in the tumors were also assessed. Stromal CD163⁺ infiltration was significantly associated with the tumor location in the tongue, and stromal and tumoral CD68⁺ and CD163⁺-infiltrating TAMs were more abundant in nonsmokers and non-alcohol-drinkers. Strikingly, this study uncovers an inverse relationship between CD68⁺ and CD163⁺ TAMs and CSC marker expression (NANOG and SOX2) in OSCC. High infiltration of CD163⁺ TAMs in both tumor and stroma was strongly and significantly correlated with the absence of NANOG expression. Moreover, infiltration of both CD68⁺ and CD163⁺ TAMs was also significantly associated with high tumor expression of PD-L1. Our results suggest that there is a link between TAM infiltration and immune escape in OSCC.

This study investigates the relevance of tumor-infiltrating lymphocytes (TILs) in oral squamous cell carcinoma (OSCC). Immunohistochemical analysis of stromal/tumoral CD4⁺, CD8⁺ and FOXP3⁺ TILs is performed in 125 OSCC patients. Potential relationships with the expression of tumoral PD-L1 and cancer stem cell (CSC) markers (NANOG, SOX2, OCT4, Nestin and Podoplanin (PDPN)) are assessed. CD4⁺ and CD8⁺ TILs are significantly associated with smoking and alcohol habits. CD4⁺ and CD8⁺ TILs show an inverse relationship with NANOG and SOX2 expression, and FOXP3⁺ TILs is significantly correlated with Nestin and PDPN expression. High infiltration of CD4⁺ and CD8⁺ TILs and a high tumoral CD8⁺/FOXP3⁺ ratio are significantly associated with tumors harboring positive PD-L1 expression. Infiltration of stromal/tumoral FOXP3⁺ TILs and a low stromal CD8⁺/FOXP3⁺ ratio are significantly associated with better disease-specific survival. Multivariate analysis reveals that the stromal CD8⁺/FOXP3⁺ TILs ratio is a significant independent prognostic factor. Regarding OSCC patient survival, the CD8⁺/FOXP3⁺ TILs ratio is an independent prognostic factor. TILs may act as biomarkers and potential therapeutic targets for OSCC.

Potentially malignant oral lesions, mainly leukoplakia, are common. Malignant transformation varies widely, even in the absence of histological features such as dysplasia. Hence, there is a need for novel biomarker-based systems to more accurately predict the risk of cancer progression. The pluripotency transcription factor SOX2 is frequently overexpressed in cancers, including oral squamous cell carcinoma (OSCC), thereby providing a link between malignancy and stemness. This study investigates the clinical relevance of SOX2 protein expression in early stages of oral carcinogenesis as a cancer risk biomarker, and also its impact on prognosis and disease outcome at late stages of OSCC progression. SOX2 expression was evaluated by immunohistochemistry in 55 patients with oral epithelial dysplasia, and in 125 patients with OSCC, and correlated with clinicopathological data and outcomes. Nuclear SOX2 expression was detected in four (7%) cases of oral epithelial dysplasia, using a cut-off of 10% stained nuclei, and in 16 (29%) cases when any positive nuclei was evaluated. Univariate analysis showed that SOX2 expression and histopathological grading were significantly associated with oral cancer risk; and both were found to be significant independent predictors in the multivariate analysis. Nuclear SOX2 expression was also found in 49 (39%) OSCC cases, was more frequent in early tumor stages and N0 cases, and was associated with a better survival. In conclusion, SOX2 expression emerges as an independent predictor of oral cancer risk in patients with oral leukoplakia. These findings underscore the relevant role of SOX2 in early oral tumorigenesis rather than in tumor progression.