The research protocol was approved by the Institutional Review Board of West China University Hospital, Sichuan University (Chengdu, China). All patients provided written informed consent and agreed to be involved in the present study. Between September 2011 and April 2012, 39 patients (34 male, 5 female; mean age, 46.9; age range 22–69) with pathologically confirmed NPC from West China University Hospital were enrolled in the present study. Each study patient was evaluated by flexible fiberoptic endoscopic examination, magnetic resonance imaging scans of the head and neck, chest X-ray, bone scan and ultrasound of the abdomen prior to treatment. X-ray computed tomography scans of the chest/abdomen were performed when clinically indicated. The histology of the tumor was evaluated according to the World Health Organization classification (13). Tumors were staged by Tumor-Node-Metastasis classification and clinical staging according to the American Joint Committee on Cancer 2009 cancer staging classification (13). NPC patient characteristics are presented in Table I.

A total of 20 healthy individuals (15 females and 5 males) were recruited for the present study (mean age, 24.8 years; range, 20–34 years).

Blood and saliva samples were collected from the healthy volunteers and from the patients with NPC prior to treatment. Vein blood (1 ml) was collected into a sterile EDTA-containing vacutainer tube from each patient. Samples were then centrifuged at 400 × g and 4°C for 10 min. The serum was collected, aliquoted and stored at −80°C until use. At least 1 ml saliva was collected using a sterile container. Samples were then centrifuged at 400 × g and 4°C for 10 min. The serum was collected, aliquoted and stored at −80°C until use.

Titers of antibodies directed at NA1 [immunoglobulin A (IgA)] in the saliva and serum samples were determined in duplicate by ELISA using commercial kits (anti-EBNA1 ELISA kit; catalog no. 03-01-60402) purchased from Zhongshan Bio-Tech Co., Ltd. (Guangzhou, China). Samples with antibody levels higher than the detection limits were diluted and measured again. In the Laboratory of Molecular and Translational Medicine, West China Second University Hospital, Sichuan University, Chengdu, China), the intra-assay and inter-assay coefficients of variation were ≤10% for all assays. The experiments were conducted by following the manufacturer's suggested procedures. Two wells of blank controls (sample dilution buffer), two negative controls and two positive controls (as provided in the kit) were included on each plate. Briefly, diluted saliva and serum samples were added to each well (100 µl in each well) of a 96-well plate. Following incubation at room temperature (RT) for 2 h on a microplate agitator rotating at 25 g, the plate was then washed with 1X PBS wash buffer with 0.05% Tween-20. Horseradish peroxidase (HRP)-conjugated anti-human IgA antibody from the kit (100 µl) was added to each well. Following incubation at RT for 20 min, the plate was then washed with 1X PBS wash buffer. Color development was conducted by addition of 100 µl tetramethylbenzidine substrate solution and incubation at RT for 10 min. Once the reaction was stopped, optical absorbance (OA) was immediately read on a spectrophotometer (Infinite M200; Tecan Trading AG, Männedorf, Switzerland) using 450 nm as the primary wavelength and 630 nm as the secondary wavelength. OA value at a wavelength of 450/630 nm for each test was determined.

The immunohistochemical (IHC) staining was performed on paraffin-embedded nasopharyngeal tissue sections from 39 NPC patients. As shown in goat anti-human CD80 polyclonal antibody (1:100; IgG; catalog no. AF140; R&D Systems, Inc., Minneapolis, MN, USA), goat anti-human CD86 polyclonal antibody (1:100; IgG; catalog no. AF-141-NA; R&D Systems, Inc.), mouse anti-human CD4 monoclonal antibody (1:100; IgG1; catalog no. MAB379; R&D Systems, Inc.), rabbit anti-human CD3 polyclonal antibody (1:100; IgG; catalog no. ab5690; Abcam, Cambridge, UK), mouse anti-human CD19 monoclonal antibody (1:100; IgG2a, κ; catalog no. ab31947; Abcam), mouse anti-human human leukocyte antigen-antigen D related (1:100; HLA-DR) monoclonal antibody (IgG1; catalog no. ab20181; Abcam), mouse anti-human IgA monoclonal antibody (1:300; IgG1; catalog no. GTX17839; GeneTex, Inc., Irvine, CA, USA) or mouse anti-human EBNA1 antibody (1:300; IgG1; catalog no. GTX40777; GeneTex) were used as the primary antibodies for IHC staining. A negative control was set up using isotype control of goat serum (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China), mouse IgG1, rabbit IgG, mouse IgG2a or mouse κ as the substitute for the primary antibody, respectively, according to the source of the primary antibody. All antibodies were diluted in PBS containing 0.1% bovine serum albumin (Roche Applied Science, Rotkreuz, Switzerland) and 0.01% sodium azide. Briefly, 4-µm tissue sections were deparaffinized for 15 min in xylene, hydrated in gradient ethyl alcohol of 100, 95, 85, 80 and 75%, and then incubated with 3% hydrogen peroxidase for 10 min at room temperature to block endogenous peroxidase. Following antigen retrieval in 0.1 mol/l citrate buffer (pH 6.0) for 15 min at 95°C in a pressure cooker, sections were cooled to RT and washed three times with PBS, and then incubated with the aforementioned diluted primary antibodies at 4°C overnight. Subsequently, sections were rewarmed to RT for 30 min and washed three times with PBS and then incubated with HRP-conjugated goat anti-rabbit/mouse secondary antibody (1:100; catalog no. ab6720; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) for 40 min at 37°C. Diaminobenzidine was used at room temperature for 3 min for color development. Finally, the sections were counterstained with Mayer's hematoxylin and mounted in Permount (ZSGB-BIO, Beijing, China). Subsequently, light microscopy observation (magnification, ×4,000; 5 fields of view) was performed. The reactivity of cell type and location in the immunostained tissues were determined.

OA values at wavelength 450/630 nm of NA1 IgA in saliva and serum samples are expressed as the mean value ± standard deviation. The statistical significance of the findings was assessed by one-way analysis of variance using Prism 5 from GraphPad Software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 1, antibody titers directed at NA1 were elevated in the saliva and serum of the majority of NPC patients, and anti-NA1 antibody titer in the serum was significantly higher than that of in the saliva of NPC patients (P<0.01). The cut-off value was defined as A_450/630 of 0.3. Compared with that of the saliva of the healthy control, the anti-NA1 antibody titer in the saliva of the NPC patients was significantly higher (P<0.01). Anti-NA1 antibody titer was determined to be negative in the saliva of the 20 healthy volunteers; however, 43.6% (17/39) of the patients with NPC were positive for the antibody. Furthermore, 74.4% (29/39) of the patients with NPC were positive for anti-NA1 antibodies in the sera.

As shown in and Fig. 2, HLA-DR, CD80, CD86, CD3, CD4, CD19, IgA and EBNA1 antibodies were used as the primary antibodies for IHC staining. A negative control was set up using an isotype control of goat serum, mouse IgG1, rabbit IgG, mouse IgG2a or mouse κ as the substitute for each primary antibody, respectively, according to the source of the primary antibody.

When the control antibodies were used, no positive signals were noted on tissue sections from patients with NPC. In infiltrating cells, the expression of HLA-DR, CD80 and CD86 was detected at the membrane of dendritic cells, the expression of CD3 and CD4 was detected at the membrane of T lymphocytes, the expression of CD19, IgA was detected at the membrane of B lymphocytes and the expression of EBNA1 protein was highly expressed at the membrane of the tumor cells in NPC tissue specimens (Fig. 2).