Ferrari et al. BMC Cancer 2012, 12:208 http://www.biomedcentral.com/1471-2407/12/208 RISH) can be considered the gold standard. The PCR was performed in a mixture containing 10 pmol of each primer (forward: 5’-GACGAGGGGCCAGGTACA GG-3’; reverse: 5’-GCAGCCAATGCTTCTTGGACGTT TTTGG-3’), 0.2 mmol/L dNTPs, 1.5 mmol/L MgCl2, and 1.25 U TaqGold polymerase (Applied Biosystems, Foster City, California, US) in a total volume of 50 μL. The thermocycling conditions consisted of 40 cycles at 94 °C for 10 min, 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, followed by a final extension phase at 72 ° C for 7 min (Thermal cycler ABI 9700, Applied Biosys- tems, Foster City, California, US). Appropriate positive and negative controls were used in each experiment. Ten microlitres of PCR products were run on a QIAxcel Sys- tem (Qiagen, Hilden, Germany) and visualised using QIAxcel BioCalculator Sofware. The expected size of the PCR product was 241 bp. Plasma samples EDTA plasma samples were collected, centrifuged at 1000 x g for 15 minutes, and then stored at −80 °C until further processing. EBV DNA copy numbers were evalu- ated before treatment, 1–2 weeks after CRT, and every six months during the follow-up period or in the case of a suspected or documented recurrence. The plasma samples were thawed and centrifuged at 20,000 x g for five minutes, and plasma DNA was extracted using a QIAamp DNA Blood MiniKit (Qiagen, Hilden, Germany). About 200–400 μL of each sample per col- umn was used for DNA extraction, and the exact amount of extracted plasma was documented fin order to calculate the target DNA concentration. Fifty microli- tres of distilled water was used to elute the DNA from the extraction column. The plasma concentrations of EBV DNA were measured by means of a real-time quantitative PCR assay of the Bam HI-W region of the EBV genome. The sequences of the forward and reverse primers were re- spectively 5'-CCCAACACTCCACCACACC-3' and 5'- TCTTAGGAGCTGTCCGAGGG-3'. A dual fluorescence- labelled oligomer, 5'-(FAM)CA CACACTACACACAC CCACCCGTCTC(TAMRA)-3', was used as a probe. The real-time quantitative PCR assay and reaction set-up proce- dures have been previously described in detail. The Bam HI-W region showed a strong correlation with the EBNA-1 PCR [28]. Real-time quantitative PCR was performed using a Rotor Gene Q (Qiagen, Hilden, Germany) analyser, and the plasma concentrations of EBV DNA were expressed as the number of copies of the EBV genome per millilitre of plasma. Values of <350 copies/mL were considered normal on the basis of the analytical sensitivity of the test. Plasma EBV DNA levels were also evaluated in 20 control samples taken from patients with head-and-neck cancers other than NPC, none of which had values of >350 copies/ml. Page 3 of 7 Informed consent was obtained from all of the patients, and the study protocol was approved by our local Ethics Committee. Statistical analysis The aim of the study was to explore the relationship be- tween plasma EBV DNA concentrations and progression- free survival (PFS). Response to treatment and overall sur- vival (OS) were also assessed. PFS was calculated from the first day of treatment to the date of relapse. The patients who had not experienced a recurrence by the analysis were censored at their last disease assessment. OS was defined as the time from the first day of treatment to the date of death due to any cause. At the time of analysis, the patients who were not reported as having died were censored at the date they were last known to be alive. Induction chemotherapy was started within three days of the date of diagnosis in all patients. Survival curves were estimated using the Kaplan- Meier method. Plasma EBV DNA concentrations were ana- lysed as both continuous and categorical variables (<350 or >350 copies), and compared using the Mann–Whitney rank-sum test. Given the exploratory nature of the study, no formal sample size calculation was made. All of the stat- istical tests were two-sided, and a p-value <0.05 was con- sidered statistically significant. The analyses were carried out using SAS software, version 9.1 (SAS Institute, Cary, North Carolina, US). Results Thirty-six consecutive Italian patients with pathologic- ally confirmed stage IIb-IVb NPC referred to the Oncol- ogy Department of San Paolo Hospital, Milan (Italy), were enrolled between October 2005 and August 2009. According to the American Joint Committee on Cancer (AJCC) classification, 7th edition (33), 11 patients (30.6%) were in stage IIb, 16 (44.4%) in stage III, six (16.7%) in stage IVa, and three (8.3%) in stage IVb. Hist- ology was type IIa in two cases (5.6%), and type IIb in the remaining 34 (94.4%). Twenty-six patients (72.2%) were males, and the median age was 52 years (range 29–58). Table 1 shows the characteristics of the enrolled patients. Before treatment, tumoral tissue from all of the patients contained EBV DNA. All but one patient had a plasma EBV DNA concentration of >350 copies/mL, with a median value of 4,701 (range 349–270,000) and an interquartile range (IQR) of 1,462-9,625. The stage IIb patients had significantly lower EBV DNA copy numbers (p = 0.007) than those in stages III-IV patients (median 2,420; IQR 820–3,939 vs median 7,520 and IQR 3187–12,745). Thirty-one patients (86.1%) received the PF induction chemotherapy regimen and five the TPF regimen. At the end of CRT, a complete response (CR) was achieved by 30 patients (83.3%, 95% CI 67.2- 93.6%) and a partial