Multiplex immunohistochemistry for immune profiling of HPV-associated head and neck cancer

Human Papilloma Virus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) is clinically distinct from HPV-negative HNSCC, and as such requires differential therapeutic approaches. Accumulating evidence indicates a significant linkage between the immune response within the tissue and pathogenesis of HPV-associated HNSCC. To further elucidate immune-related signatures in HPV-associated HNSCC, we performed multiplex histological analysis in de-identified tissue microarray sections including HPV-positive (n = 21), HPV-negative (n = 17), and normal oropharynx (n = 8). Following immunohistochemistry (IHC) for CD45, CD3, CD8, Foxp3, T-bet, GATA-3, RORgT, CD20, CD56, CD68, MHC class II, CSF1R, CD66b, tryptase, CD83, DC-SIGN, PD-1, and PD-L1, the cell intensity per mm² ratio/composition, localization were quantitatively evaluated. The HPV-status was confirmed by HPV16/18 polymerase chain reaction and IHC for p16^INK4a. IHC for p16 or EpCAM were utilized for defining tumor region. Infiltration of T cell populations including CD45⁺CD3⁺CD8⁺ T cells (P < 0.01), CD45⁺CD3⁺CD8⁻Foxp3⁺ regulatory T cells (P < 0.05) and CD45⁺CD3⁺CD8⁻Foxp3⁺T-bet⁺ Th1 cells (P < 0.01), CD45⁺CD20⁺CD3⁻B cells (P < 0.05), CD45⁺CD68⁺CD163⁻ macrophages (P < 0.001), and CD45⁺Tryptase⁺ mast cells (P < 0.01) was significantly higher in the HPV-positive group than in the HPV-negative group. CD8/CD68 ratio of HPV-positive tumor was higher than that of HPV-negative tumor (P < 0.05), and the highest CD163⁻CD68⁺/CD163⁺CD68⁺ ratio was observed in the intra-tumor region of HPV-positive tumors. High PD-L1 expression on CD68⁺CD163⁺ macrophages and MHC class II⁺CD83⁺ dendritic cells was intensively observed in the intra-tumor region of the HPV-positive group while maturation of dendritic cells assessed by CD83/DC-SIGN ratio was significantly higher in the peritumoral stroma than intra-tumor regions (P < 0.05), indicating distinct immune regulatory mechanisms between intra and peritumoral regions. These signatures provide further evidence of anti-tumor immunity against HPV-positive head and neck cancer, and potentially lead to personalized treatment including immunomodulatory therapeutic targets specialized for the HPV/immune status.