Methylation analysis of plasma Epstein-Barr virus (EBV) DNA in EBV-associated diseases

Wai Kei Jacky Lam ^1,2,3 Peiyong Jiang ^1,2,3 Kwan Chee Allen Chan ^1,2,3 Wenlei Peng ^1,2 Huimin Shang ^1,2 Macy May Sze Heung ^1,2 Suk Hang Cheng ^1,2 Haiqiang Zhang ^1,2 On Yee Olivia Tse ^1,2 Radha Raghupathy ^3,4 Brigette Buig Yue Ma ^3,4 Edwin Pun Hui ^3,4 Anthony Tak Cheung Chan ^3,4 John Kong Sang Woo ⁵ Rossa Wai Kwun Chiu ^1,2,3 Yuk Ming Dennis Lo ^1,2,3

¹Department of Chemical Pathology, The Chinese University of Hong Kong, Hong Kong
²Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong
³State Key Laboratory of Translational Oncology, The Chinese University of Hong Kong, Hong Kong
⁴Department of Clinical Oncology, The Chinese University of Hong Kong, Hong Kong
⁵Department of Otorhinolaryngology, Head and Neck Surgery, The Chinese University of Hong Kong, Hong Kong

Epstein-Barr virus (EBV) is associated with a number of diseases. Circulating EBV DNA has been explored as a biomarker in different EBV-associated diseases, in particular malignancies.Currently, it is not known whether the methylation profiles of circulating EBV DNA would be different in patients with different EBV-associated diseases. We performed methylation analysis of plasma DNA samples from patients with nasopharyngeal carcinoma (NPC), EBV-associated lymphoma and infectious mononucleosis by targeted bisulfite sequencing. Different plasma EBV DNA methylation profiles were observed in different diseases. These results have significant implications in the development of methylation analysis of plasma EBV DNA for NPC diagnostics. To explore the clinical utility of methylation analysis for NPC screening, we analyzed the plasma EBV DNA methylome of 33 NPC and 160 non-NPC subjects from a prospective cohort of more than 20,000 subjects recruited for the screening of NPC. Differential methylation patterns in the EBV genome were observed between NPC and non-NPC subjects. When analysis of such differential methylation patterns was combined with the analyses of the fractional concentration and size of plasma EBV DNA fragments, population screening of NPC could be performed with a positive predictive value of 35.7%, compared to the current polymerase-chain-reaction-based protocol (11.0%). These results thus demonstrate the potential clinical application of methylation analysis of plasma EBV DNA for screening NPC.