HPV-positive status associated with inflamed immune microenvironment and improved response to anti-PD-1 therapy in head and neck squamous cell carcinoma

Abstract

Chemotherapy and radiotherapy predominantly improve the clinical outcomes of patients with human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC). Whether this superiority goes on when treated with immune checkpoint inhibitors is still unclear. This study sought to determine the predictive value and potential mechanisms of HPV status for the treatment of programmed cell death 1 (PD-1)/ligand 1(PD-L1) inhibitors. We conducted an integrated analysis of the relationships between HPV status and PD-L1, tumor mutation burden (TMB) and inflammation-related immune cells and molecules, based on the analysis of repository databases and resected HNSCC specimens. The pooled analysis of overall survival (OS) and objective response rate (ORR) suggested that HPV-positive patients benefited more from PD-1/PD-L1 inhibitors than HPV-negative patients (OS: hazard ratio (HR) = 0.71, p = 0.02; ORR: 21.9% vs 14.1%, odds ratio (OR) = 1.79, p = 0.01). Analysis of public databases and resected HNSCC specimens revealed that HPV status was independent of PD-L1 expression and TMB in HNSCC. However, HPV infection significantly increased T-cell infiltration, immune effector cell activation and the diversity of T-cell receptors. Notably, HPV-positivity correlated with increased immune cytolytic activity and a T-cell-inflamed gene expression profile. This work provides evidence that HPV status can be used to predict the effectiveness of PD-1 inhibitors in HNSCC, independently of PD-L1 expression and TMB, and probably results from an inflamed immune microenvironment induced by HPV infection.

Figure 1

Forest plots of hazard ratios (HRs) for overall survival (a) and odds ratios (ORs) for objective response rate (b) from six clinical trials, comparing human papillomavirus (HPV)-positive with HPV-negative head and neck squamous cell carcinoma (HNSCC) patients treated with programmed cell death 1 (PD-1)/ligand 1(PD-L1) inhibitors. Pooled HRs and ORs were computed using the fixed-effects model. Pos, positive; Neg, negative.

Figure 2

HPV-positive status was independent of PD-L1 expression in patients with head and neck squamous cell carcinoma (HNSCC). (a) Quantitative analysis of PD-L1 RNA expression from RNA-seq profiles in patients with HPV-positive and -negative HNSCC based on The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO: GSE40774) database. (b) Correlation analysis of HPV viral titers and PD-L1 expression based on the TCGA cohort. (c) PD-L1 levels were analyzed in seven HNSCC cell lines with confirmed HPV status based on GSE62027 RNA-seq profiles. (d) Quantitative analysis of PD-L1 protein expression derived from reverse phase protein array according to HPV status. (e) Immunohistochemical (IHC) analysis of PD-L1 protein expression based on HPV status in a cohort of 130 resected HNSCC patients. TC, tumor cell; IC, immune cell; Pos, positive; Neg, negative.

Figure 3

HPV infection did not directly impact tumor mutation profiles of head and neck squamous cell carcinoma (HNSCC) patients. (a–c) Quantitative analyses of tumor mutational burden (TMB), neoantigens count, and copy number alteration (CNA) in HPV-positive and -negative tumors based on the TCGA cohort. (d–f) Correlation analyses of HPV viral titers and TMB, neoantigen count, and CNA based on the TCGA cohort. (g,h) Quantitative analyses of TMB and CNA in HPV-positive and -negative HNSCC based on the MSK-IMPACT cohort.

Figure 4

HPV infection promoted T-cell infiltration and T-cell receptor (TCR) diversity. (a) Quantitative analysis of CD8A RNA expression from RNA-seq profiles in patients with HPV-positive and -negative head and neck squamous cell carcinoma (HNSCC) based on the TCGA and GSE40774 cohorts. (b) Correlation analysis of HPV viral titers and CD8A expression based on the TCGA cohort. (c) Representative images of immunostaining of PD-L1, p16, and CD8 in serial sections of HNSCC tumors. (d) Immunohistochemical (IHC) analysis of CD8 protein expression comparing HPV-positive with HPV-negative status in a cohort of 130 resected HNSCC patients. (e) Quantitative analysis of lymphocyte infiltration signature score in patients with HPV-positive and -negative HNSCC from the TCGA database. (f) Gene set enrichment analysis (GSEA) revealed upregulation of the T-cell receptor signaling pathway in the HPV-positive group compared with the HPV-negative group. (g,h) Quantitative analysis of TCR diversity and TCR richness in patients with HPV-positive and -negative HNSCC from the TCGA database. TCGA, The Cancer Genome Atlas; NES, normalized enrichment score.

Figure 5

HPV-positive status correlated with increased cytotoxicity and T cell-inflamed gene expression profiles (GEPs). (a–c) Gene set enrichment analysis (GSEA) revealed upregulation of the IL2 signaling pathway and downregulation of the IL6 and TGFB1 pathways in the HPV-positive group compared with the HPV-negative group. (d) Quantitative analysis of cytolytic activity (CYT) in patients with HPV-positive and -negative head and neck squamous cell carcinoma (HNSCC) based on the TCGA and GSE40774 cohorts. (e) Correlation analysis of HPV viral titers and CYT based on the TCGA cohort. (f,h) Heatmap depicting 18 genes representative of T cell-inflamed GEP correlated to the HPV status of the corresponding HNSCC tissues based on the TCGA and GSE40774 cohorts. (g,i) Quantitative analysis of GEP scores in patients with HPV-positive and -negative HNSCC based on the TCGA and GSE40774 cohorts. TCGA, The Cancer Genome Atlas; NES, normalized enrichment score.