# Blind Brush Biopsy: Quantification of Epstein–Barr Virus and Its Host DNA Methylation in the Detection of Nasopharyngeal Carcinoma

## TL;DR

This study develops a non-invasive blind brush biopsy method to detect nasopharyngeal carcinoma using DNA methylation markers from Epstein-Barr virus and host genes.

## Contribution

The study introduces a novel diagnostic method combining EBV and host DNA methylation markers for NPC detection using blind brush sampling.

## Key findings

- A single EBV methylation marker (BILF2) achieved 80.22% sensitivity and 98.44% specificity in NPC detection.
- Combining BILF2 with host marker IMPA2 increased sensitivity to 84.62% without reducing specificity.
- Methylation markers were almost exclusively detected in NPC samples, with minimal presence in controls.

## Abstract

Background: The nasopharyngeal brush sampling can effectively collect samples from the nasopharynx. The blind brush sampling does not require the guidance of endoscopy, which is favorable for implementation and dissemination in the community. This study explored methylation markers for nasopharyngeal carcinoma (NPC) at both Epstein–Barr virus (EBV) and its host genome levels, aiming to construct a blind brushing diagnostic method. Methods: EBV DNA capture and methylation sequencing and GEO Illumina 450K methylation array data were used respectively for the discovery of EBV and host methylation markers. The diagnostic method was built in training cohort (n = 347) and validated in an independent validation cohort (n = 155). Results: A total of 1 EBV methylation marker (BILF2) and 6 host methylation markers (ITGA4, IMPA2, ITPKB, PI9, AMIGO2, and VAV3) were identified. Both EBV and host methylation markers were almost exclusively detected in NPC samples, with negligible detection in control samples. In validation cohort, the diagnostic method that included only the EBV BILF2 marker showed a sensitivity and specificity of 80.22% and 98.44%, respectively. When combining the EBV-derived marker BILF2 with the host-derived marker IMPA2, the diagnostic method’s sensitivity increased to 84.62%, while the specificity remained unchanged (IDI = 4.4%, P = 0.0419). Conclusion: Overall, the blind nasopharyngeal brushing diagnostic method, combining EBV and host methylation markers, showed great potential in NPC detection and could promote its application in nonclinical screening of NPC.