Analysis of PD-L1 promoter methylation combined with immunogenic context in pancreatic ductal adenocarcinoma

Abstract

Despite the successful application of programmed cell death ligand 1 (PD-L1)-blocking strategies in some types of cancers and well-established prognostic indicators in pancreatic ductal adenocarcinoma (PDAC), the biological and clinical implications of the methylation status of PD-L1/PD-L2 in PDAC remain largely unknown. Therefore, this study aimed to explore the biological role of PD-L1/PD-L2 methylation and its association with clinicopathological features, clinical outcomes, and the immune microenvironment by analyzing the data on PD-L1/PD-L2 methylation and mRNA expression in PDAC cohorts obtained from the Cancer Genome Atlas and International Cancer Genome Consortium. The correlation between PD-L1 promoter methylation and PD-L1 expression and survival was further validated in an independent validation cohort (Peking Union Medical College Hospital [PUMCH] cohort) using pyrosequencing and immunohistochemistry. These results demonstrated that hypomethylation of the PD-L1 promoter was strongly associated with upregulated PD-L1 expression and shorter overall survival in PDAC. Multivariate Cox regression analyses revealed that the PD-L1 promoter methylation was an independent prognostic factor. PD-L1 promoter hypomethylation and high expression were related to aggressive clinical phenotypes. Moreover, both PD-L1 and PD-L2 methylation correlated with immune cell infiltration and the expression of immune checkpoint genes. PD-L1 promoter methylation status was further validated as an independent prognostic biomarker in patients with PDAC using the PUMCH cohort. The prognostic significance of PD-L1 promoter methylation was more discriminative in tumors with perineural/lymphovascular invasion and distant metastasis than in those without perineural/lymphovascular invasion and distant metastasis. In summary, the methylation status of the PD-L1 promoter is a promising biomarker for survival outcomes, immune infiltration, and the potential immune benefits of immunotherapy in PDAC.

Keywords: Epigenetic biomarker; Methylation; PD-L1; PD-L2; Pancreatic ductal adenocarcinoma; Survival.

Fig. 1

PD-L1/PD-L2 methylation status was correlated with corresponding CD274/PDCD1LG2 expression in PDAC. A–B. The correlation of cg19724470 and cg02823866 with CD274 expression in the TCGA cohort; C–E. The correlation of cg14133064, cg14374994, cg14351952, and cg07211259 with PDCD1LG2 expression in the TCGA cohort. p values < 0.05 were marked in red

Fig. 2

PD-L1 promoter methylation (cg19724470) predicted overall survival in PDAC. A–L. Kaplan–Meier survival analysis of cg19724470, cg02823866, cg14351952, cg14374994, cg07211259, and cg14133064 for overall survival of patients from the TCGA and ICGC cohorts, respectively. The best cutoff was applied in each Kaplan–Meier analysis. The blue line represents the hypomethylation group, and the red lines represent the hypermethylation group. M–N. Multivariate Cox analysis of cg19724470 methylation and the clinicopathological factors significant in the univariate Cox analysis (p < 0.05) from the TCGA and ICGC cohorts, respectively. p values < 0.05 were marked in red

Fig. 3

PD-L1/PD-L2 expression and corresponding methylation probes were correlated with immune cell infiltration and immunotherapy response in PDAC. A–B. The correlation heatmap of PD-L1/PD-L2 expression and related methylation probes with 34 types of immune cells in the TCGA and ICGC cohorts, respectively. The correlation coefficients and statistical significance are shown in each grid; C–D. Relative enrichment scores of 14 immunotherapy response-related pathways in hypermethylation and hypomethylation subgroups of cg19724470 in the TCGA and ICGC cohorts, respectively. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns, not significant

Fig. 4

CD274/PDCD1LG2 expression and corresponding methylation probes were correlated with the expression of ICGs in PDAC. A. The correlation heatmap of PD-L1/PD-L2 expression and related methylated CpG sites with 65 detected ICGs in the TCGA cohort; B–E. The correlation of cg02823866 or cg07211259, and CD274/PDCD1LG2 expression with immune checkpoint molecules PDCD1, CTLA4, LAG3, and TIGIT, respectively, in the TCGA cohort. p values < 0.05 were marked in red

Fig. 5

cg19724470 methylation and PD-L1 expression were associated with overall survival in PUMCH cohort. A. Kaplan–Meier survival analysis of cg19724470 methylation; B. Kaplan–Meier survival analysis of PD-L1 TPS; C. The representative IHC slides of PD-L1 in the hypermethylation and hypomethylation subgroups of PUMCH cohort; D. Multivariate Cox analysis of cg19724470 methylation, PD-L1 TPS, and the clinicopathological factors significant in the univariate Cox analysis (p < 0.05); E. Kaplan–Meier survival analysis of PD-L1 TPS in the hypermethylation subgroup; F. Kaplan–Meier survival analysis of PD-L1 TPS in the hypomethylation subgroup. PD-L1, programmed cell death ligand 1; TC, tumor cell. p values < 0.05 were marked in red

Fig. 6

PD-L1 promoter methylation exhibited modification effect induced by PNI and LVI in the PUMCH cohort. A. Results of multivariate Cox analysis of PD-L1 promoter methylation per crucial clinical feature subgroups. p values < 0.05 were bolded; B–K. Kaplan–Meier curves of overall survival categorized by PD-L1 promoter methylation status in patients with histologic grade (B, C), PNI (D, E), LVI (F, G), T stage (H, I), N stage (J, K), and M stage (L, M). PNI, perineural invasion; LVI, lymphovascular invasion; HR, hazard ratio; CI, confidence interval. p values < 0.05 were marked in red