Characterization of extracellular secreted Epstein-Barr virus BART microRNAs as serological markers for nasopharyngeal carcinoma diagnosis and treatment

Abstract

Nasopharyngeal carcinoma (NPC) exhibits a distinct geographical and ethnic distribution. It is recognized that 75-90% of NPC patients have already developed local or regional spread at diagnosis, which hampers effective treatment and results in a poor prognosis. It is essential to characterize more sensitive and specific biomarkers for NPC screening of high risk individuals and for differentiation of treatment options upon diagnosis. One of the hallmarks of NPC is the ubiquitous association with Epstein-Barr virus (EBV). In NPC cells, EBV expresses only very essential viral proteins, but more than 20 BART microRNAs (miRNAs) at abundant levels. This study characterized NPC specific BART miRNAs through a comprehensive analysis of EBV BART microRNA expression profiles in EBV infected cells, including latency I and III EBV infected BL cells, EBV transformed LCLs, EBV-harbouring NPC cells and non-cancerous nasopharyngeal cells. We further characterized the BART miRNAs secreted into culture supernatants and determined that BART-3, BART-7 and BART-13 miRNAs are regularly secreted into the extracellular environment, implicating these BART miRNAs as potential serological biomarkers for use in NPC screening. Analysis of plasma specimens obtained from NPC patients and healthy and non-NPC tumor patient controls found levels of both BART-7 and BART-13 to be significantly higher among NPC patients, with elevated levels being particularly apparent among patients with advanced disease. Levels of BART-3 were indistinguishable between NPC and control groups. We further confirmed that BART-7 and BART-13 are NPC associated. Analysis of 41 NPC patients before and after successful radiotherapy treatment showed that BART-7 and BART-13, but not BART-3, were diminished after treatment. Receiver Operating Characteristic (ROC) curve analysis combining BART-7 and BART-13 levels produces a 90% predictive value for the presence of NPC. These results indicate that BART-7 and BART-13 may be useful new serological biomarkers for diagnosis of NPC and prediction of treatment efficacy.