HNSCC Gene Expression Subtypes, Including a HPV Subtype, Demonstrate Differential Immune Cell and Biomarker Associations

Abstract

Human papilloma virus (HPV) RNA expression in combination with a gene expression signature of 144 genes was used to classify head and neck squamous cell carcinoma (HNSCC) into 5 distinct subtypes. The subtypes are investigated for differential associations with clinical features, immune infiltration, and immune marker expression. Using previously published Bindea et al. immune cell gene signatures (24 in total) and the TCGA Head & Neck cancer gene expression dataset (HNSCC n=520), we examined immune cell expression in relation to 5 HNSCC gene expression subtypes (atypical, classical, basal, mesenchymal, and HPV subtype). The HPV tumor subtype was determined by alignment of RNAseq with HPV sequences and by evaluation of its gene expression profile. Signatures of multiple immune cells as well as single immune-biomarkers, (CTLA4, PDCD1(PD-1), and CD274(PD-L1) were examined across the 5 subtypes. Differential gene expression was examined using the Kruskal-Wallis test. Immune cell signature associations with tumor subtype and with CD274 expression were evaluated using linear regression. Survival-based associations were evaluated with cox proportional hazard models. Immune cell expression was significantly different across the subtypes (Tcells KW P=1.7e-17) and in general was highest in HPV positive tumors. The classical expression subtype demonstrated lower Tcell immune expression. CD274 (PD-L1) expression was less variable across the subtypes but lowest expression was again seen in the classical subtype (KW P=3.7e-06). Mutation burden varied across the subtypes with the highest mutation burden observed in the atypical subtype and the lowest mutation burden in the HPV tumors (KW P=1.7e-10). Subtype and HPV status were more strongly associated with immune expression than CD274 (PD-L1) expression (median F-test P-value and adjusted R-squared were 2.0e-16 and 0.14 for subtype versus 6.7e-07 and 0.04 for CD274). Cox models with adjustment for stage suggested higher immune cell expression is associated with better survival, and most strongly in the HPV tumor subtype including T gamma delta cells HR 0.2 (95% CI 0.06-0.68) and T cells HR 0.33 (95% CI 0.14-0.79). Biologic subtypes of HNSCC reveal key differences in immune cell expression, which were not always correlated with CD274 (PD-L1) expression. Evaluation of subtypes as potential biomarkers for immunotherapy response in HNSCC should be investigated.