Ferrari et al. BMC Cancer 2012, 12:208 http://www.biomedcentral.com/1471-2407/12/208 (type I); non-keratinizing squamous cell carcinoma, differ- entiated (type IIa); non-keratinizing squamous cell carcin- omas, undifferentiated (type IIb); basaloid squamous cell carcinomas (type III) [15]. Epidemiological studies have shown that the gamma– herpes Epstein-Barr virus (EBV) plays a major role in the pathogenesis of NPCs as they are endemic in areas where EBV infection is also endemic [16-18]. Potentially EBV-induced carcinogenesis depends on the expression of a set of oncogenes: in particular, latent membrane protein 1 (LMP1) plays a role in preventing apoptosis and activating a number of signalling pathways such as nuclear factor k-B (NF-kB), mitogen-activated protein (MAP) kinases, and phosphoinositol-3-kinase (PI3K), thus favouring cell motility and suppressing immuno- genic responses [19,20]. Following the demonstration of EBV in histological samples of NPCs [21,22] and the presence of high titres of IgA antibody against a viral capside antigen (VCA) [23,24], it has been recently demonstrated that real-time quantitative polymerase chain reaction (PCR) can easily detect cell-free EBV DNA in the serum of NPC patients [25-29]. Previous studies have found different percen- tages of patients whose plasma was positive for EBV DNA and, sometimes, a significant proportion of patients with a low viral load, possibly due to differences in PCR methods [30]. The great majority of patients in endemic countries have high circulating levels of EBV DNA at diagnosis [31], and the higher the level, the greater the risk of recurrence. Furthermore recurrent/ metastatic disease can be heralded by an increase in plasma EBV DNA levels during follow-up [26]. There are few data concerning the role of EBV DNA in Western populations, but an Italian study has demon- strated similar behaviour in a low-incidence region in which EBV DNA may be of prognostic value [32]. The aim of this prospective study was to evaluate EBV DNA levels before treatment and during follow-up in a cohort of Western patients with loco-regionally advanced NPCs. Methods Untreated patients with pathologically confirmed stage IIb-IVb NPC [33] were considered eligible for study in- clusion. The pre-treatment evaluation included a phys- ical examination, the assessment of performance status, a complete blood cell count with differential, a biochem- ical profile, the quantification of plasma EBV DNA copy numbers, MR imaging, and the measurement of index lesions. The baseline imaging techniques included PET and computed tomography (CT) in the case of suspected metastasis. The patients were treated by means of induction chemo- therapy followed by concomitant chemo-radiotherapy (CRT). Induction chemotherapy consisted of three cycles Page 2 of 7 of cisplatin (CDDP) 100 mg/m2 i.v. on day 1 and 5- fluorouracil (5-FU) 1000 g/m2/day administered as a con- tinuous intravenous infusion on days 1–4 (PF regimen), or three cycles of CDDP 75 mg/m2 i.v. on day 1, docetaxel 75 mg/m2 i.v. on day 1, and 5-FU 750 mg/m2/day adminis- tered as a continuous intravenous infusion on days 1–4 (TPF regimen), both repeated every three weeks. The chemotherapy was administered via a central venous cath- eter in an outpatient setting. During the TPF regimen, prophylactic antibiotics were given between the cycles. During RT, CDDP was administered as a 100 mg/m2 intravenous infusion 60 minutes before radiation on days 1, 22 and 43. The patients were hydrated with mannitol and/or furosemide in order to maintain a high urine output. The intravenous anti-emetics given before CDDP included granisetron 3 mg and dexamethasone 8 mg. Three-dimensional conformal RT was delivered in sin- gle daily fractions of 2 Gy on five days a week without interruptions. The planned total dose to the tumour and involved lymph nodes was 70 Gy in 35 fractions. Tumour response was assessed by means of a clinical examination, endoscopy and MR using the response evaluation criteria in solid tumours (RECIST) [34] at the end of chemotherapy and 8–12 weeks after the comple- tion of CRT. The patients were followed up by means of a clinical examination every two months during the first year, every three months for the following two years, and every six months thereafter. Salvage treatment after a re- lapse that was not eligible for surgery or re-irradiation consisted of four cycles of carboplatin AUC 5 and pacli- taxel 175 mg/m2 on day 1 q 21. Tissue samples Formalin-fixed paraffin-embedded (FFPE) tissue blocks were retrieved from the archives of the Path- ology Unit. Three 10 μm thick sections from each block were collected in a 1.5 mL tube, dewaxed with xylene, dehydrated with ethanol, and incubated over- night in a digestion buffer containing 50 mM Tris, pH 8, 5 mM EDTA, pH 8, 0.5% Tween20, and 10 μL proteinase K (20 mg/ml). After digestion, total DNA was purified using DNA QIAamp DNA MiniKit (Qiagen, Hilden, Germany) spin columns as indicated by the manufacturer. The quality of the DNA was tested by means of the multiplex amplification of four fragments of 100, 200, 300 and 400 bp as previously described [35], and was considered acceptable in the presence of at least the 100, 200 and 300 bp ladders. For each sample, 5 μL of total DNA was tested for the presence of the EBV DNA IR3 region (EBNA1) using previously described primers [36]. The EBNA 1 PCR is a simple method to detect EBV DNA even if in situ hybridization for EBV-encoded RNA (EBER-