Plasma cfDNA VILL gene methylation as a diagnostic marker for nasopharyngeal carcinoma

Currently, the non-invasive diagnostic methods for nasopharyngeal carcinoma (NPC) continue to grapple with the challenge of low sensitivity. The hypermethylation of tumor suppressor genes is an established early event in NPC pathogenesis. Consequently, we conducted whole-genome methylation sequencing on plasma cell-free DNA (cfDNA) from six NPC cases and four healthy controls, integrating Illumina Human Methylation 450 K microarray data from the GEO database comprising six NPC cases and six samples of non-cancerous nasopharyngeal tissue (NP). As result, we screened only one CpG island associated with cell type-specific regulation within the candidate tumor suppressor gene VILL (Vilin Like), which exhibits specific methylation patterns in NPC. We validated our findings using 25 pairs of NPC and NP samples from GEO, alongside 9,736 pan-cancer tissues from TCGA and 656 healthy human leukocyte samples sourced from GEO through methylation microarray analysis. Based on this, we designed a methylation-specific qPCR (qMSP) system for the VILL gene, and then tested it on 192 primary NPC and 154 NC plasma samples. The new qMSP system when compared with EBV DNA qPCR revealed a sensitivity for primary NPC of 80.2% vs.81.3% (78.8% vs.54.5% for early-stage NPC), and a specificity of 100% vs. 93.5%. Notably, employing a combined methodology further enhanced sensitivity to 94.8%, including a sensitivity rate of 90.9% for early-stage NPC diagnosis. Therefore, VILL methylation assessment combined with EBV DNA detection presents a promising avenue for non-invasive diagnosis of NPC, particularly beneficial for early detection.