Identification of functional methylated CpG loci in PD-L1 promoter as the novel epigenetic biomarkers for primary gastric cancer.

Gastric cancer (GC) is considered one of the most lethal malignancies worldwide due to poor prognosis. Aberrant methylation has been demonstrated to be involved in PD-L1 dysregulated expression in human cancers and possesses a great value as a diagnostic biomarker. Given that, in this study, we investigated the methylation status of PD-L1 as a promising biomarker in primary gastric tumors and identified functional CpG loci undergoing aberrant methylation through tumorigenesis of GC. PD-L1 methylation was initially evaluated in-silico using TCGA-STAD dataset. Pearson's correlation analysis was further employed to identify the most significant functional methylated CpG loci of PD-L1 gene in TCGA-STAD patient cohort. Methylation status and its correlation with PD-L1 expression were also validated using q-MSP and qRT-PCR in a set of internal samples, including 25 paired primary gastric tumors and adjacent normal tissues. The obtained results from TCGA-STAD showed that PD-L1 is significantly hypermethylated through gastric tumorigenesis, mostly in two CpG loci overlapping with cg19724470 and cg15837913 probes. Besides, PD-L1 DNA methylation was negatively correlated with PD-L1 expression in tumor samples. Furthermore, hypermethylation of cg19724470 and cg15837913 regions was validated in primary gastric tumors compared to adjacent normal samples. Also, ROC curve analysis illustrated the high diagnostic value of PD-L1 methylation for early detection of GC (AUC = 0.8110). In conclusion, the findings of this study suggested that PD-L1 expression is regulated by methylation in functional CpG loci and its methylation could be considered as a valuable diagnostic target for GC.