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.

Immunohistochemical analysis of stromal/tumoral CD20+ B lymphocytes was performed in 125 OSCC patients. Correlations with immune profiles CD4+, CD8+, and FOXP3+ tumor-infiltrating lymphocytes (TILs), tumoral PD-L1, and stem-related factors NANOG and SOX2 were assessed, and also associations with clinical data and patient survival. There was a strong positive correlation between the infiltration of CD20+ B lymphocytes and other immune profiles (i.e., CD4+, CD8+, and FOXP3+ TILs, and CD68+ and CD163+ macrophages) both in stroma and tumor nests. Strikingly, CD20+ TILs were inversely correlated with NANOG/SOX2 expression. Stromal CD20+ TILs were significantly associated with T classification and second primary tumors. A stratified survival analysis showed that tumoral CD20+ TILs were significantly associated with prognosis in male and younger patients, with tobacco or alcohol consumption, high tumoral CD8+ TILs, low tumoral infiltration by CD68+ macrophages, positive PD-L1 expression, and negative NANOG/SOX2. Multivariate Cox analysis further revealed clinical stage and tumoral CD20+ TILs independently associated with disease-specific survival (HR = 2.42, p = 0.003; and HR = 0.57, p = 0.04, respectively). In conclusion, high CD20+ TIL density emerges as an independent good prognostic factor in OSCC, suggesting a role in antitumor immunity. This study also uncovered an inverse correlation between CD20+ TILs and CSC marker expression.

Zinc finger AN1-type containing 4 (ZFAND4) has emerged as a promising prognostic marker and predictor of metastasis for patients with oral squamous cell carcinoma (OSCC). However, further validation is fundamental before clinical implementation. Hence, this study evaluated the expression pattern of ZFAND4 protein expression by immunohistochemistry using an independent cohort of 125 patients with OSCC, and correlations with the clinicopathologic parameters and disease outcome. Remarkably, ZFAND4 expression, while negligible in normal epithelium, exhibited two distinct expression patterns in tumors that did not overlap. A gross granular staining was characteristic of the undifferentiated cells at the invasive front of tumors, whereas the most differentiated cells located at the center of the tumor nests showed diffuse non-granular staining. ZFAND4 staining was higher in undifferentiated than in differentiated areas of tumors. High ZFAND4 expression in differentiated cells was significantly associated to well-differentiated (p = 0.04) and non-recurrent tumors (p = 0.04), whereas ZFAND4 expression in undifferentiated cells correlated with tumor location (p = 0.005). No correlations between the ZFAND4 expression and patient survival were found. These data question the clinical relevance of ZFAND4 expression as a prognostic biomarker in OSCC, and also reveal distinct ZFAND4 expression patterns depending on the differentiation areas of tumors that should be evaluated separately.

PurposeThe aim of this study was to investigate the prognostic significance of preoperative inflammatory markers in peripheral blood of patients with oral squamous cell carcinoma (OSCC), and to establish correlations with the infiltrate of macrophages and lymphocytes in the local immune tumor microenvironment (TME).Materials and MethodsNeutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), lymphocyte-monocyte ratio (LMR), and systemic immune-inflammation index (SII) were retrospectively evaluated in a cohort of 348 OSCC patients, and correlated with overall (OS) and disease-specific survival (DSS). Immunohistochemical analysis of tumoral and stromal infiltration of CD8+, CD4+, FOXP3+ and CD20+ lymphocytes and CD68+ and CD163+ macrophages was performed in a subset of 119 OSCC patient samples, and correlations further assessed.ResultsNLR, SII, and LMR were significantly associated with a poorer OS in univariate analysis; however, only NLR remained a significant independent predictor in the multivariate analysis (HR = 1.626, p = 0.04). NLR and SII were inversely and significantly correlated with stromal infiltration of CD8+, CD4+, and CD20+ lymphocytes. Moreover, a significant correlation between LMR was also found to significantly associate with stromal infiltration of CD8+, CD4+, and CD20+ lymphocytes, stromal CD68+ and CD163+ macrophages, and also tumoral infiltration of CD4+ and CD20+ lymphocytes.ConclusionsPreoperative NLR, SII, and LMR may serve as valuable systemic markers to predict OSCC patient survival, with NLR emerging as an independent predictor of poor OS. Moreover, strong significant correlations were exclusively observed between systemic inflammatory markers and the local stromal infiltration of lymphocytes in the TME.

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