High-Sensitivity PD-L1 Staining Using Clone 73-10 Antibody and Spatial Transcriptomics for Precise Expression Analysis in Non-Tumorous, Intraepithelial neoplasia, and Squamous Cell Carcinoma of Head and Neck

Purpose : Although immune checkpoint inhibitors (ICIs) have improved the prognosis of head and neck squamous cell carcinoma (HNSCC), certain patients remain ineligible based on PD-1 immunohistochemistry (IHC). We aimed to evaluate the PD-L1 ( CD274 ) expression using highly sensitive clone 73-10 and spatial transcriptomics (ST) analysis to elucidate the role of PD-L1 in HNSCC and thus potentially expand the pool of eligible patients. Methods: Immunohistochemical staining of 73-10, CD3, CD4, and CD8 were performed in 94 HNSCC clinical samples along with paired adjacent squamous intraepithelial neoplasm (SIN) and normal oral mucosa (NOM) samples. The 73-10 positivity was evaluated using a tumor cell score ≥ 1%, and the results were analyzed against clinicopathological features including CD4 ⁺ and CD8 ⁺ tumor-infiltrating lymphocytes (TILs), and clinical outcomes. Furthermore, ST and PD-L1 related pathway analysis was performed in 6 paired HNSCC, SIN and NOM samples. Results: 73-10 detected-PD-L1 positivity was high in HNSCC (79%) than SIN (10%) and NOM (2%), and correlated with high CD4 ⁺ TILs, and also independent prognostic factor of OS, DSS, and PFS (all p < 0.05). ST analysis revealed the upregulated distribution of CD274 correlated with 73-10 positivity. Pathway analysis identified significantly upregulation of CD274 and C D4 in HNSCC than SIN and NOM, and HIF-1α and IFN-γ as key regulators of PD-L1 expression in HNSCC. Conclusion: PD-L1 might be an effective ICI target, and the 73-10 IHC demonstrates high sensitivity in detecting PD-L1 ( CD274 ) in HNSCC, offering immunological and prognostic insights that can aid in improving patient selection for ICI therapy.