The Immune Checkpoint Receptor CD96: A Local and Systemic Immune Modulator in Oral Cancer?

SIMPLE SUMMARY: As immune checkpoint inhibitor (ICI) therapy against PD1 is only efficient in a small proportion of OSCC patients, identification of further checkpoints might improve therapy response by enabling combination ICI treatment. The aim of this study was to analyze the gene- and protein-expression of the checkpoint CD96 in tissue and peripheral blood of OSCC patients compared to healthy controls, while also checking for associations to histomorphological parameters. Patients and controls were analyzed by real-time quantitative polymerase chain reaction and by immunohistochemistry. CD96 expression in tumor tissue and peripheral blood of OSCC patients is differentially regulated. Tumor tissue showed a significant upregulation of CD96 expression. mRNA and protein expression correlated significantly. In peripheral blood of OSCC patients a significant downregulation of CD96 was observable. CD96 expression correlated with other immune checkpoints. CD96 might be a relevant immune checkpoint and needs further investigation especially in the context of immunotherapy. ABSTRACT: Background: As immunotherapy of oral squamous cell carcinomas (OSCCs), using PD1 inhibitors, is only efficient in a small proportion of patients, additional immune checkpoints need to be identified as potential therapeutic targets. There is evidence that a blockade of CD96 might positively affect the anti-tumor immune response. The aim of this study was to analyze the gene and protein expression of CD96 in the tissue and peripheral blood of OSCC patients compared to healthy controls, while also checking for potential associations with a differential expression to the histomorphological parameters. In addition, possible correlations with the expression of PD1 and PD-L1 as well as the macrophage markers CD68 and CD163 should be tested to obtain further insights into the potential effectiveness of combined checkpoint blockage. Material and Methods: For real-time quantitative polymerase chain reaction (RT-qPCR), a total of 183 blood and tissue samples, divided into a patient and a control group, were included. Additionally, 141 tissue samples were examined by immunohistochemistry (IHC). The relative expression differences between the groups were calculated using statistical tests including the Mann–Whitney U test and AUC method. The Chi-square test was used to determine whether CD96 overexpression in individual samples is associated with malignancy. Correlation analysis was performed using the Spearman correlation test. Results: There was a significant CD96 mRNA and protein overexpression in the OSCC group compared to the controls (p = 0.001). In contrast, CD96 mRNA expression in the peripheral blood of the OSCC patients was significantly lower compared to the control group (p = 0.007). In the Chi-square test, the OSCC tissue samples showed a highly significant upregulation of CD96 mRNA expression (p < 0.001) and protein expression (p = 0.005) compared to the healthy mucosa. CD96 mRNA and protein expression correlated significantly (p = 0.005). In addition, there was a significant positive correlation of CD96 expression with PD1 (p ≤ 0.001), PD-L1 (p ≤ 0.001), and CD163 (p = 0.006) at the mRNA level. Conclusions: CD96 expression in the tumor tissue and peripheral blood of OSCC patients is differentially regulated and appears to be a relevant immune checkpoint.