PD-L1 (CD274) and PD-L2 (PDCD1LG2) promoter methylation is associated with HPV infection and transcriptional repression in head and neck squamous cell carcinomas

Background: DNA methylation of the immune checkpoint gene PD-L1 has recently been shown to be associated with PD-L1 mRNA expression in various malignancies. This study aimed to investigate the association of PD-L1 and PD-L2 methylation with mRNA expression, immune cell infitration, protein expression and human papilloma virus (HPV) infection in head and neck squamous cell carcinoma (HNSCC) patients. Results: DNA methylation of PD-L1 and PD-L2 correlates inversely with mRNA expression (PD-L1: p <= 0.002; PD-L2: p <= 0.014). Methylation of specific CpG-sites of both PD-L1 and PD-L2 were further significantly associated with HPV infection in the TCGA cohort. Immune cell infiltrates correlated significantly with PD-L1 and PD-L2 methylation. In the validation cohort, PD-L1 protein expression was associated with PD-L1 hypomethylation (p = 0.012). Conclusions: DNA methylation of PD-L1 and PD-L2 is associated with transcriptional silencing and HPV infection in HNSCCs. Additional studies are warranted to test PD-L1 and PD-L2 methylation as predictive biomarkers for response to immunotherapies (e.g. pembrolizumab and nivolumab) that target the PD-L1/PD-L2/PD-1 immune checkpoint axis. Materials and Methods: PD-L1 and PD-L2 promoter methylation and its mRNA expression were analyzed based on Infinium HumanMethylation450 BeadChip and RNA-Seq (both Illumina, Inc.) data in a representative HNSCC patient cohort (n = 528) enrolled by The Cancer Genome Atlas (TCGA) Research Network. A validation cohort consisting of 168 HNSCC patients treated at the University Hospital Bonn was analyzed regarding PD-L1 and PD-L2 promoter methylation by means of methylation-specific quantitative real-time PCR. PD-L1 protein expression in the validation cohort was quantified via immunohistochemistry (PD-L1 antibody clone 22C3, Dako/Agilent Technologies, Inc.).