Ferrari et al. BMC Cancer 2012, 12:208 http://www.biomedcentral.com/1471-2407/12/208 Table 1 Patients' characteristics Number of patients Median age (years) Range Sex – n (%) Male WHO histology – n (%) AJCC stage - n (%) IIa IIb IIb III IVa IVb 0 1 WHO Performance status Positive EBV-DNA WHO = World Health Organization. AJCC = American Joint Committee on Cancer. EBV = Epstein-Barr Virus. 36 52.0 29-58 26 (72.2) 2 (5.6) 34 (94.4) 11 (30.6) 16 (44.4) 6 (16.7) 3 (8.3) 33 (91.7) 3 (8.3) 36 (100) response (PR) by the remaining six. EBV DNA levels had returned to below the threshold limit in all of the complete responders and four of the partial responders; the two partial responders with still high EBV DNA levels underwent a biopsy that failed to reveal the pres- ence of tumour cells. No further treatment was given to the patients with a PR. After a median follow-up of 36 months (IQR 24–48 months), seven patients had experienced recurrent disease (4 loco-regional and 3 metastatic), one of whom had died. Three-year PFS and 3-year OS were respectively 78.2% and 97.1% Figure 1. EBV DNA load monitoring seemed to be a reliable molecular marker for predicting recurrence as five of the seven patients who relapsed (sensitivity 71.3%, 95% CI 29.0%-96.3%) had EBV DNA copy numbers that were much higher than the limit of detection (900 and 2,135 copies/mL in two patients with loco-regional relapse; and 9,185, 15,290 and 4,398,000 copies/mL respectively in three patients with metastases). The loco-regional re- lapse in the remaining two patients was not heralded by an increase in EBV DNA levels (Table 2) but by appear- ance of latero-cervical lymphadenopathy confirmed by node biopsy in one case (12 mm nodal relapse) and both nodal and nasopharyngeal biopsy in the second (respect- ively 11 mm and 17 mm; loco-regional relapse). MR im- aging confirmed the diagnoses. The patient with the highest EBV DNA load died of disseminated disease 13 months after diagnosis. Page 4 of 7 None of the 29 patients who did not experience a re- currence had EBV DNA levels above the cut-off value during the follow-up, which suggests a specificity of 100% (95%CI 88.1-100%). The overall accuracy of plasma EBV DNA levels in truly detecting recurrence was 94.4% (34/36, 95%CI 81.3-99.3). Median plasma EBV DNA concentrations were 270 copies/mL in the patients without recurrent disease and 2,135 copies/mL in the seven experiencing recurrence (p = 0.037). Pre-treatment EBV DNA concentrations sig- nificantly correlated with PFS at univariate analysis (p = 0.036), but not after adjusting for stage (p = 0.051). Discussion It has been demonstrated that NPCs are associated with EBV DNA infection as the virus infects the epithelial cells promoting the activation of proliferation signalling [37]. Tumour cells release EBV DNA during treatment, and plasma EBV DNA levels correlate with tumour vol- ume and TNM stage [27,38-40]. Quantitative real-time PCR can detect circulating EBV DNA, and the concen- tration of short cell-free EBV DNA fragments can pre- dict recurrence and survival. It has been shown that almost all NPC patients have variable pre-treatment EBV DNA copy numbers, and that the highest values correlate with a poor prognosis [41]. In the case of a CR, pre-treatment levels become undetectable after RT or CRT, whereas some patients with a PR, stable disease or progression tend to maintain high EBV DNA levels. It has been reported that patients receiving standard CRT can be stratified into subgroups on the basis of their pre- and post-treatment EBV DNA levels. Five-year OS and 5-year relapse-free survival are significantly better in the groups with low or high pre-treatment and undetect- able post-treatment levels than in those with still detect- able levels at the end of treatment [41]. In our study, 97% of the patients had pre-treatment levels of >350 copies/mL that significantly correlated with tumour stage (lower values in stage IIb than stages III-IV). According to Leung et al. [40] high pre- treatment concentrations during early-stage NPCs are associated with the risk of distant metastases, and Lin et al. [38] found that pre-treatment plasma EBV DNA concentrations were lower in patients with local recur- rences than in those with distant metastases (1,311 vs 4,253 copies/mL) even if the difference was not statisti- cally significant (p = 0.37). They also described 10 patients (10.1%) with detectable EBV DNA levels one week after completing RT, seven of whom experienced a distant relapse, which reflects the importance of un- detectable plasma EBV DNA levels in maintaining a disease-free state. Hou et al. [39] found that both pre- and post- treatment EBV DNA concentrations were significantly