Spatial transcriptomic analysis reveals significant differences in tumor microenvironment in HPV-dependent and HPV-independent vulvar squamous cell carcinoma.

Objective: Vulvar squamous cell carcinoma (VSCC) can be either HPV-dependent (HPVd) or HPV-independent (HPVi). HPVd VSCC typically occurs in younger women, has a more favorable prognosis, and develops from high-grade squamous intraepithelial lesions (HSIL). HPVi VSCC predominantly affects older women and arises within areas of chronic inflammation, particularly lichen sclerosis (LS). We utilized sequencing-based spatial transcriptomics to explore gene expression in a cohort of patients with HPVi and HPVd VSCC.

Methods: We analysed gene expression in distinct areas (SCC, inflammation, LS, HSIL) from four early-stage VSCC cases (two HPVi, two HPVd) using the 10× Genomics Visium spatial transcriptomics platform. Cell-specific type expression was inferred using CIBERSORTx.

Results: 28,183 Visium spots were detected; each contained an estimated 20-50 cells. Reads per spot ranged from 9903 to 68,527. More genes were upregulated in HPVd (N = 601) than HPVi (N = 72) with distinct differences in Keratin and Collagen genes between etiologies. Gene expression was strikingly similar between SCC and adjacent inflammatory areas, regardless of etiology. IL-17 signaling was upregulated in HPVd samples. Surprisingly, CIBERSORTx inferred significantly more CD45+ cells in HPVi tissues than HPVd, especially CD4+ resting memory and follicular helper T cells in SCC areas. Immune cells moved from resting states in the pre-invasive tissues to activated states in the SCC and peri-tumoral inflammatory areas.

Conclusions: This study represents the first application of spatial transcriptomics in VSCC, with significantly more immune cells identified in HPVi SCC than in HPVd SCC. These data will act as a baseline for future studies.