Spatial Profiles of Intratumoral PD-1(+) Helper T Cells Predict Prognosis in Head and Neck Squamous Cell Carcinoma

BACKGROUND: Functional interactions between immune cells and neoplastic cells in the tumor immune microenvironment have been actively pursued for both biomarker discovery for patient stratification, as well as therapeutic anti-cancer targets to improve clinical outcomes. Although accumulating evidence indicates that intratumoral infiltration of immune cells has prognostic significance, limited information is available on the spatial infiltration patterns of immune cells within intratumoral regions. This study aimed to understand the intratumoral heterogeneity and spatial distribution of immune cell infiltrates associated with cell phenotypes and prognosis in head and neck squamous cell carcinoma (HNSCC). METHODS: A total of 88 specimens of oropharyngeal squamous cell carcinoma, categorized into discovery (n = 38) and validation cohorts (n = 51), were analyzed for immune contexture by multiplexed immunohistochemistry (IHC) and image cytometry-based quantification. Tissue segmentation was performed according to a mathematical morphological approach using neoplastic cell IHC images to dissect intratumoral regions into tumor cell nests versus intratumoral stroma. RESULTS: Tissue segmentation revealed heterogeneity in intratumoral T cells, varying from tumor cell nest-polarized to intratumoral stroma-polarized distributions. Leukocyte composition analysis revealed higher ratios of T(H)1/T(H)2 in tumor cell nests with higher percentages of helper T cells, B cells, and CD66b(+) granulocytes within intratumoral stroma. A discovery and validation approach revealed a high density of programmed death receptor-1 (PD-1)(+) helper T cells in tumor cell nests as a negative prognostic factor for short overall survival. CD163(+) tumor-associated macrophages (TAM) provided the strongest correlation with PD-1(+) helper T cells, and cases with a high density of PD-1(+) helper T cells and CD163(+) TAM had a significantly shorter overall survival than other cases. CONCLUSION: This study reveals the significance of analyzing intratumoral cell nests and reports that an immune microenvironment with a high density of PD-1(+) helper T cells in tumoral cell nests is a poor prognostic factor for HNSCC.