Spatial Transcriptome Analysis of B7-H4 in Head and Neck Squamous Cell Carcinoma: A Novel Therapeutic Target for Anti-Immune Checkpoint Inhibitors

Head Neck Pathol. 2025 Jun 30;19(1):78. doi: 10.1007/s12105-025-01815-w.

Abstract

Purpose: A significant proportion of patients with head and neck squamous cell carcinoma (HNSCC) are ineligible for immune checkpoint inhibitors (ICIs) because of low programmed cell death protein-ligand 1 (PD-L1) expression. The therapeutic potential of B7-H4 (VTCN1) was investigated using immunohistochemistry (IHC) and spatial transcriptomics (ST).

Methods: IHC analysis of B7-H4, PD-L1, CD3, CD4, and CD8 was performed using a tissue microarray [94 HNSCC, 94 adjacent squamous intraepithelial neoplasia (SIN), and 69 adjacent normal oral mucosa (NOM) samples]. B7-H4 and PD-L1 expression levels were assessed using tumor cell score (TC; positive, TC > 1%), immune cell score, and combined positive score. ST was performed on six HNSCCs with paired SINs and NOMs to confirm the expression and distribution of B7-H4 (CTVN1), PD-L1 (CD274), CD4 (DC4A), and CD8 (CD8).

Results: In HNSCCs, TCs revealed a mutually exclusive B7-H4/PD-L1 expression pattern in 55% of samples (p < 0.05). B7-H4 positive TCs were more frequent in HNSCCs (79%) than in SINs (10%) and NOMs (2%). ST analysis confirmed mutually exclusive VTCN1/CD274 upregulation in 83% of samples (n = 6) and demonstrated co-localization of B7-H4 protein and VTCN1 in IHC-positive areas. B7-H4 was significantly correlated with low-CD8+ T-cell infiltration (n = 94, p = 0.009), and CD8A mRNA was down-regulated in the VTCN1+ area compared with that in the VTCN1+ area.

Conclusion: B7-H4 is a promising antibody-drug conjugate target in ICI-resistant HNSCC. IHC combined with TCs enabled the reliable assessment of B7-H4, given its co-localization with VTCN1 in IHC-positive areas and association with low-CD8+ T-cells.

Keywords: Antibody–drug conjugate; B7-H4; Immune checkpoint inhibitor; PD-L1; Squamous cell carcinoma.

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • B7-H1 Antigen / metabolism
  • Biomarkers, Tumor* / analysis
  • Biomarkers, Tumor* / genetics
  • Female
  • Gene Expression Profiling
  • Head and Neck Neoplasms* / drug therapy
  • Head and Neck Neoplasms* / genetics
  • Head and Neck Neoplasms* / immunology
  • Head and Neck Neoplasms* / pathology
  • Humans
  • Immune Checkpoint Inhibitors / pharmacology
  • Immune Checkpoint Inhibitors / therapeutic use
  • Male
  • Middle Aged
  • Squamous Cell Carcinoma of Head and Neck* / drug therapy
  • Squamous Cell Carcinoma of Head and Neck* / genetics
  • Squamous Cell Carcinoma of Head and Neck* / immunology
  • Squamous Cell Carcinoma of Head and Neck* / metabolism
  • Squamous Cell Carcinoma of Head and Neck* / pathology
  • V-Set Domain-Containing T-Cell Activation Inhibitor 1* / biosynthesis
  • V-Set Domain-Containing T-Cell Activation Inhibitor 1* / genetics
  • V-Set Domain-Containing T-Cell Activation Inhibitor 1* / metabolism

Substances

  • Immune Checkpoint Inhibitors
  • V-Set Domain-Containing T-Cell Activation Inhibitor 1
  • VTCN1 protein, human
  • Biomarkers, Tumor
  • B7-H1 Antigen
  • CD274 protein, human