# Methylation-specific qPCR for the EBV C promoter to quantify EBV methylation

## TL;DR

A new methylation-specific PCR assay for the EBV C promoter can detect methylation levels in EBV-associated tumors and patient samples.

## Contribution

A novel methylation-specific qPCR assay (MSPCP) was developed and validated for quantifying EBV C promoter methylation in clinical and biological samples.

## Key findings

- MSPCP showed high-level C promoter methylation (94–100%) in EBV-associated tumors but not in lymphoid hyperplasia.
- High EBV methylation was detected in plasma cell-free DNA from patients with EBV-associated Hodgkin lymphoma.
- Low or no EBV methylation was found in saliva from a general population, consistent with virion DNA.

## Abstract

Epstein-Barr Virus (EBV) is a ubiquitous virus associated with a variety of diseases including cancers. Evidence has emerged that the C promoter is methylated in many EBV-associated malignancies, whereas in free virion DNA it is unmethylated. We have developed and evaluated a methylation-specific PCR assay for the EBV C Promoter (MSPCP) that can be applied to human biological specimens to quantify EBV methylation.

Two sets of methylation-specific primers were designed to anneal to bisulfite-converted DNA sequences with 3 CpGs in the forward primer binding site, and 2 CpGs in the reverse primer binding site. We evaluated this method in synthetic oligonucleotides, DNA extracted from cell lines, virion supernatants, and a variety of clinical specimens. EBV methylation of Cp, as measured by MSPCP, was validated with two orthogonal methods in select samples.

In contrived samples, this method had a linear range between 0–100% methylation. Application of this assay to DNA extracted from 11 formalin-fixed paraffin-embedded biopsy specimens showed high-level C promoter methylation in EBV-associated tumors (94–100%) but not in EBV-associated lymphoid hyperplasia. High-level EBV methylation was also detected in cell-free DNA extracted from the plasma of 13 patients with EBV-associated Hodgkin lymphoma. In contrast, EBV methylation was either not-detected, or detected at very low levels, in saliva from 25 adults in a general university population consistent with the presence of virion DNA.

MSPCP is a simple, rapid and accurate method that characterizes the methylation status of the EBV C promoter, which may be useful in a variety of research and clinical settings.

The online version contains supplementary material available at 10.1186/s13027-025-00702-x.