The immune microenvironment of HPV-positive and HPV-negative oropharyngeal squamous cell carcinoma: a multiparametric quantitative and spatial analysis unveils a rationale to target treatment-naïve tumors with immune checkpoint inhibitors

J Exp Clin Cancer Res. 2022 Sep 20;41(1):279. doi: 10.1186/s13046-022-02481-4.


Background: Immune checkpoint inhibitors (ICI) are approved for treatment of recurrent or metastatic oropharyngeal head and neck squamous cell carcinoma in the first- and second-line settings. However, only 15-20% of patients benefit from this treatment, a feature increasingly ascribed to the peculiar characteristics of the tumor immune microenvironment (TIME).

Methods: Immune-related gene expression profiling (GEP) and multiplex immunofluorescence (mIF) including spatial proximity analysis, were used to characterize the TIME of 39 treatment-naïve oropharyngeal squamous cell carcinomas (OPSCC) and the corresponding lymph node metastases. GEP and mIF results were correlated with disease-free survival (DFS). HPV-positive tumors disclosed a stronger activation of several immune signalling pathways, as well as a higher expression of genes related to total tumor-infiltrating lymphocytes, CD8 T cells, cytotoxic cells and exhausted CD8 cells, than HPV-negative patients. Accordingly, mIF revealed that HPV-positive lesions were heavily infiltrated as compared to HPV-negative counterparts, with a higher density of T cells and checkpoint molecules. CD8+ T cells appeared in closer proximity to tumor cells, CD163+ macrophages and FoxP3+ cells in HPV-positive primary tumors, and related metastases. In HPV-positive lesions, PD-L1 expression was increased as compared to HPV-negative samples, and PD-L1+ tumor cells and macrophages were closer to PD-1+ cytotoxic T lymphocytes. Considering the whole cohort, a positive correlation was observed between DFS and higher levels of activating immune signatures and T cell responses, higher density of PD-1+ T cells and their closer proximity to tumor cells or PD-L1+ macrophages. HPV-positive patients with higher infiltration of T cells and macrophages had a longer DFS, while CD163+ macrophages had a negative role in prognosis of HPV-negative patients.

Conclusions: Our results suggest that checkpoint expression may reflect an ongoing antitumor immune response. Thus, these observations provide the rationale for the incorporation of ICI in the loco-regional therapy strategies for patients with heavily infiltrated treatment-naïve OPSCC, and for the combination of ICI with tumor-specific T cell response inducers or TAM modulators for the "cold" OPSCC counterparts.

Keywords: Gene expression profile; Head and neck squamous cell carcinoma; Human papillomavirus; Immunotherapy; Multiplex immunofluorescence; Oropharyngeal carcinoma; Sex; Tumor microenvironment.

MeSH terms

  • B7-H1 Antigen / genetics
  • Carcinoma, Squamous Cell* / drug therapy
  • Carcinoma, Squamous Cell* / genetics
  • Forkhead Transcription Factors
  • Head and Neck Neoplasms*
  • Humans
  • Immune Checkpoint Inhibitors / pharmacology
  • Immune Checkpoint Inhibitors / therapeutic use
  • Oropharyngeal Neoplasms* / drug therapy
  • Oropharyngeal Neoplasms* / metabolism
  • Papillomavirus Infections* / complications
  • Programmed Cell Death 1 Receptor / metabolism
  • Spatial Analysis
  • Squamous Cell Carcinoma of Head and Neck / drug therapy
  • Tumor Microenvironment


  • B7-H1 Antigen
  • Forkhead Transcription Factors
  • Immune Checkpoint Inhibitors
  • Programmed Cell Death 1 Receptor