Can the co-localization of CD8+, PD-1, PD-L1 AND PD-L2 patterns provide guidance in clinical outcomes of patients with head and neck cutaneous squamous cell carcinoma?

• The co-localization of PD-1/PD-L1 could be a promising tool for prognostic evaluation. • The first study to assess co-localization of CD8, PD1, PDL1, PDL2 in cSCC. • Notable presence of CD8+T-cell infiltration in cSCC, co-localizing with PD-L1/L2. Cutaneous Squamous Cell Carcinoma (cSCC) incidence has increased almost 200% in recent years. Blockade of immune checkpoint axes is an effective way to reactivate the host anti-tumor immune response across a variety of cancer types. We aimed to assess the presence of the Programmed cell Death-1 (PD-1):PD-L1/PD-L2 axis and associated CD8+ infiltration in head and neck cSCC, establish the co-localization criteria. This retrospective, cross-sectional study included 46samples of patients with high-risk cSCC. Multiplex staining for PD-1, PD-L1, PD-L2, and CD8 was performed, and images were captured by an epifluorescence microscope. 1%, 20% and 50% expression cutoff values were used, as well as the COX regression method to calculate the Hazard Ratio (HR) and 95% Confidence Interval (95% CI). PD-1/PD-L1, PD-1/PD-L2 and PD-L1/PD-L2 co-localized in 80.4%, 45.7% and 47.8% of the patients, respectively. All patients exhibited CD8+ infiltration to some extent, with PD-1/PD-L1, PD-1/PD-L2 and PD-1/PD-L1/PD-L2 co-localized in 97.8%, 82.6% and 82.6% of the patients, respectively. There was no statistically significant relationship regarding the co-localization of immune checkpoint and the outcomes evaluated. This study has evidenced significant expression and co-localization of immune checkpoint axes in whole tissues and tumor infiltrate, emphasizing the potential application of co-localization as a valuable tool in the clinical management of cSCC. These findings provide important insights for understanding and optimizing therapeutic strategies in head and neck cSCC. 4.