Methylation Marker has a Complementary Value to Epstein-Barr Virus-Based Tests in Early Detection of Nasopharyngeal Carcinoma

ABSTRACT Undifferentiated nasopharyngeal carcinoma (NPC) is strongly linked to Epstein-Barr virus (EBV) infection. The presence of aberrant antibodies against EBV and high viral DNA load can be predictive for the disease and have been proposed for screening tools. Beside the EBV-based markers, analysis of aberrant methylation in gene promoter of tumor suppressor gene (TSG) may have potential value in identifying NPC at early stage. This study determined methylation status of multiple TSGs and evaluated whether it may improve NPC early detection using EBV-based assays. Association between EBV-encoded latent membrane protein 1 (LMP1) and methylation status was also tested. Nasopharyngeal brushings and sera were obtained from 53 NPC cases, 22 high risk individuals and 25 healthy EBV carriers. In all sera the detection of antibody against EBV was performed using EBV-IgA ELISA. EBV DNA load was measured by a quantitative light-cycler PCR on nasopharyngeal brushing samples. For methylation analysis, DNA was modified using bisulfite treatment and amplified by methylation-specific PCR (MSP) or quantitative MSP to target region within promoter of eleven TSGs. The expression of LMP1 was tested using immunohistochemistry. NPC samples demonstrated frequent methylation for individual TSGs (DAPK1 79%, CDH13 77%, DLC1 77%, RASSF1A 76%, CADM1 70%, p16 66%, WIF1 61%, CHFR 59%, RIZ1 57% and RASSF2A 29%). High risk individuals showed high frequency of four methylated genes, low methylation of two TSGs and undetectable methylation of another four TSGs. Healthy subjects had similar pattern as high risk individuals. Because not all of methylated genes allowed good discrimination between NPC and healthy individuals, a panel of markers (RASSF1A, p16, WIF1, CHFR and RIZ1) was determined and combined analysis revealed a detection rate of 98%. Quantitative analysis of MAL gene, TSG that has never been tested in NPC, showed promising result for NPC marker. The expression of LMP1 was observed to associate with methylated RASSF1A. In conclusion, methylation analysis has a complementary value to EBV-based assays for NPC risk assessment. The association of EBV oncogene to methylation status underlined the role of EBV in epigenetic event during oncogenesis. Disclosure All authors have declared no conflicts of interest.