Quantitative plasma/serum EBV DNA load by LMP2A determination in an Italian cohort of NPC patients

Abstract Background: Nasopharyngeal carcinoma (NPC) is frequently associated with Epstein–Barr virus (EBV), but little is known about the EBV DNA prevalence on peripheral blood in Western Countries, where the tumour is not endemic and its incidence is low. Objectives: To set up and evaluate an internally controlled qualitative polymerase chain reaction (PCR) followed by quantitative competitive PCR for the detection of EBV DNA in clinical specimens. To investigate whether EBV DNA load in peripheral blood was a consistent feature of Italian NPC patients. Materials and methods: A PCR assay based on latent membrane protein 2A (LMP2A) sequence amplification was chosen. Best assay conditions, sensitivity and reproducibility were determined. Sixty-four sera and 63 plasma from an Italian cohort of 39 NPC patients were analyzed. Samples from 5 patients followed up after radiotherapy were also assayed. Qualitative and quantitative β-globin amplification was performed in parallel in order to provide an independent control for amplification competence of DNA and to investigate whether EBV DNA levels could be due to intracellular EBV viral genomes from cells lysed during plasma/serum collection. Results: Twenty-five patients had undifferentiated carcinoma (UC) and 14 squamous cell carcinoma (SCC). EBV DNA has been quantified in 58 and 9% of the UC and SCC cases, respectively. No statistically significative differences were observed between the EBV DNA levels (469 vs 750 copies/ml, P =0.16) and prevalence (64 vs 57%, X 2 1 =0.22, P =0.64) in plasma and serum samples. Increased EBV viremia was found in patients with considerable extension of the primary tumour (172 vs 2250 copies/ml, low vs high tumour burden). Three UC subjects, which had detectable pre-treatment EBV DNA levels, became negative after radiotherapy. Clinical examination revealed that all had complete tumour regression. Conclusions: These PCR procedures allow an accurate and reproducible estimation of plasma/serum EBV DNA load in NPC patients living in non endemic areas, being strictly associated with UC WHO III and with tumour severity.