EBV DNA methylation profiles and its application in distinguishing nasopharyngeal carcinoma and nasal NK/T-cell lymphoma

Abstract

Background: As an oncovirus, EBV is associated with multiple cancers, including solid tumors and hematological malignancies. EBV methylation plays an important role in regulating tumor occurrence. However, the EBV methylation profiles in EBV-associated tumor tissues are poorly understood.

Results: In this study, EBV methylation capture sequencing was conducted in several different tumor tissue samples, including NPC, EBVaGC, lung LELC and parotid LELC. Besides, EBV capture sequencing and following qMSP were performed on nasopharyngeal brushing samples from NPC and nasal NKTCL patients. Our results showed that the EBV genome among different types of tumors displayed specific methylation patterns. Among the four types of tumors from epithelial origin (NPC, EBVaGC, lung LELC and parotid LELC), the most significant differences were found between EBVaGC and the others. For example, in EBVaGC, all CpG sites within 1,44,189-1,45,136 bp of the EBV genome sequence on gene RPMS1 were hyper-methylated compared to the others. Differently, significant differences of EBV CpG sites, particularly those located on gene BILF2, were observed between NPC and nasal NKTCL patients in nasopharyngeal brushing samples. Further, the methylated level of BILF2 was further detected using qMSP, and a diagnostic model distinguishing NPC and nasal NKTCL was established. The AUC of the model was 0.9801 (95% CI 0.9524-1.0000), with the sensitivity and specificity of 98.81% (95% CI 93.63-99.94%) and 76.92% (95% CI 49.74-91.82%), respectively.

Conclusions: Our study reveals more clues for further understanding the pathogenesis of EBV, and provides a possibility for distinguishing EBV-related tumor by detecting specific EBV CpG sites.

Keywords: EBV, EBV DNA methylation; EBV-associated tumor; NPC; Nasal NKTCL.

Fig. 1

Distribution of EBV CpG sites methylation levels in four tumor tissue samples. A Box plots of the average methylation density of CpG sites annotated on different phases genes. B Methylation density plot of EBV CpG sites in NPC, EBVaGC, lung LELC and parotid LELC tissue samples. The higher the ridge, the more CpG sites at that methylation level. C EBV genome circos plot. The inner circle was a scatter plot of methylation density of EBV sites in each tumor tissue, where different colors represented different types of cancer, and darker colors indicated higher levels of methylation

Fig. 2

Heatmap of EBV methylation density in four tumor tissue samples. A Methylation density heatmap of 81 EBV genes annotated with 3497 CpG loci in NPC, EBVaGC, lung LELC and parotid LELC tissues. The redder the color, the higher the methylation density, and the bluer the lower the methylation density. The genes within the red boxed area were relatively hyper-methylated; while, the genes within the blue boxed area were relatively hypo-methylated. B Methylation density heatmap of the most variable loci among these tumor tissues. In the heatmap, the black box indicated the loci with significantly higher or lower methylation levels in the corresponding tumor tissue, compared to other tumor tissues

Fig. 3

Distribution of EBV CpG sites methylation levels in NPC and nasal NKTCL samples. A The comparison of average methylation levels between NPC and nasal NKTCL nasopharyngeal brushing samples. B Methylation density plot of EBV CpG sites in NPC and nasal NKTCL nasopharyngeal brushing samples. The higher the ridge, the more CpG sites at that methylation level. C EBV genome circos plot. The inner circle was a scatter plot of methylation density of EBV sites in each nasopharyngeal brushing sample group, where different colors represented different types of cancer, and darker colors indicated higher levels of methylation

Fig. 4

Heatmap of EBV methylation density in NPC and nasal NKTCL nasopharyngeal brushing samples. A Methylation density heatmap of 80 EBV genes annotated with 3683 CpG loci in NPC and nasal NKTCL nasopharyngeal brushing samples. The redder the color, the higher the methylation density, and the bluer the lower the methylation density. B Methylation density heatmap of the most variable loci among these nasopharyngeal brushing samples

Fig. 5

Methylation of BILF2 and EBV load detected by qPCR between NPC and nasal NKTCL samples. A Comparison of EBV load results between NPC and nasal NKTCL nasopharyngeal brushing samples. B Comparison of BILF2 qMSP results between NPC and nasal NKTCL nasopharyngeal brushing samples. C ROC curve for BILF2 qMSP and EBV load results