Construction The T7 Phage CDNA Library From Nasopharyngeal Carcinoma Tissues And Screening Autoantibodies In Sera Of Patient With Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is one of the most common cancers in the Chinese and other Asian populations. It is one of the serious health problems in Southern China where an annual incidence of more than 20 cases per 100,000 was reported. Metastasis was found in 7% of the patients at the time of initial diagnosis and 20% or more developed metastasis after treatment. However, NPC is often diagnosed late due to its deep location and vague symptoms. Therefore, a diagnostic assay that can detect NPC early would greatly improve the treatment outcome of NPC.Many proteins are overexpressed in tumor tissues, which could activate the immune system to produce their antibodies. Instead of measuring the levels of these over expressed proteins, an alternative approach is to measure the circulating antibodies against tumor-associate -d proteins in the sera of patients. Some autoantibodies could have diagnostic and prognostic value, such as antibodies against BRD2 and alpha-methylacyl-CoA racemase (AMACR) for prostate cancer, antibodies against annexin XI-A and Grb2-associated protein 2 for breast cancer, and antibodies against the epithelial cell adhesion molecule (Ep-CAM) and creatine kinase B for ovarian cancer. There were also several overexpressed proteins that could be used as potential tumor markers for NPC, such as fibronectin, Mac-2 binding protein, plasminogen, inter-α-trypsin inhibitor precursor and zinc-alpha(2)-glycoprotein (ZAG). These observations strongly suggested that a list of potential autoantibody markers will greatly facilitate the development of an assay with high predictive value. Studies of the autoantibodies in NPC patients towards these NPC specific antigens could provide an effective diagnostic assay for NPC. Autoantibody signatures, as new biomarkers, may improve the early detection of nasopharyngeal carcinoma.At first, in this study, to investigate whether there are autoantibodies to NPC in the patients sera in order to find new nasopharyngeal carcinoma(NPC) biomarker, we prepared the cell plate of Epstein Barr-virus negative NPC cell line CNE1, and analyzed the differential reactions between 32 NPC patient serum and 54 normal serum by ELISA. Extracted the total protein of CNE1, and analyzed whether had the specific proteins to react with NPC serum by Western blot.The results of ELISA showed that the average of NPC patients sera antibody absorbance values(0.904±0.032) were significantly elevated above the mean of normal sera antibody absorbance values (0.736±0.028) (P<0.01). The analysis with Western blot showed there were positive bands ,and some of these were unanimously bands, but the intensity increased, and some of these were new bands compared with normal serum. These positive bands may be NPC tumor-associated antigens or NPC tumor-specific antigens. There are autoantibodies reacted with NPC in the sera of patients with NPC, and which not reacted with Epstein-Barr virus. It provided the basis to seek the tumor biomarkers in NPC serum.Secondly, to construct a T7 phage cDNA library from mixed nasopharyngeal carcinoma tissue samples of 8 patients using Novagen's OrientExpress cDNA Synthesis and Cloning Systems, and biopan the T7 phage cDNA library with sera pooled from ten NPC patients (stages II-IV) and from normal healthy donors. Twenty two phage expressed immunogenic proteins were identified through immunochemical detection and ELISA. Then we measured the levels of autoantibodies against five identified proteins in the sera of NPC patients and in the sera of healthy donors by ELISA to evaluate the sensitivity and specificity of these autoantibodies for NPC detection. Logistic regression was used to evaluate the probability of a sample to be the serum of a patient or the serum of a health donor.We constructed a T7 phage cDNA library from mixed NPC tissues, and we isolated 31 tumor-associated proteins using biopan enrichment techniques with sera from NPC patients and from healthy population. DNA sequence analysis showed that among 31 phage-displayed proteins, 22 have sequence identity with known or putative tumor-associated proteins. The results of immunochemical reactivity of patients' sera with phage expressed proteins showed enrichment in the number of immunogenic phage clones in the biopanning process and also confirmed that antibodies were present in the sera of patients but not in the sera of healthy donors.The phage expressing BMI-1 protein was isolated by screening of a mixture of nasopharyngeal carcinoma (NPC) cDNA T7 phage library and found that the antibody against BMI-1 was elevated in the sera from NPC patients. BMI-1 mRNA was over-expressed at different levels in seven NPC cell lines compared with normal nasopharyngeal epithelial cell line NP69. Histochemistry showed that patient sera were more reactive with BMI-1 than normal sera. Antibody affinity assay using sera from 40 NPC patients and 54 controls showed that BMI-1 antibody was significantly greater in patient sera than in normal controls (patient 0.791±0.025 and normal 0.488±0.042; P<0.001) and the BMI-1 autoantibody be significantly related with the progress of NPC (Benign versus LNPC P=0.001; LNPC versus MNPC P=0.047). Analysis of the results with logistic regression and receiver operating characteristics (ROC) curves showed that BMI-1 antibody was a modest marker for NPC (sensitivity 0.74 and specificity 0.73;AUC=0.8044). The showed that BMI-1 antibody as a potential marker of NPC may be rational, and could have diagnostic and prognostic value.We measured the levels of antibodies against another four phage expressed proteins and the phage expressed protein EBNA-1. The c statistic (i.e., area under the curve) of EBNA-1, which is the positive control, was 0.6110 (P=0.0120). MAGE was the most significant with a c statistic of 0.8922 (P<0.0001). The c statistic of HSP70 was 0.8435 (P<0.0001), the c statistic of CD44 was 0.7857 (P<0.0001), and the c statistic of fibronectin was 0.8091 (P<0.0001). These results showed that phage expressed proteins, HSP70, CD44, fibronectin and MAGE, as markers had greater sensitivity and specificity to predict the NPC disease than EBNA-1.There was a significant correlation between the levels of HSP70, CD44, fibronectin or MAGE antibodies with the clinical stage (P < 0.001) in the NPC groups. The autoantibody level of MAGE positively correlated with T classification: higher T classification correlated with higher autoantibody levels of MAGE(P<0.05). And the antibody levels against other three proteins partially correlated with the clinical stages of NPC. The levels of antibodies can be used to predict the clinical stages of NPC.Our studies suggested that the autoantibodies against tumor-associ -ated antigens in the sera of NPC patients could be used as a screening test for NPC and these autoantibodies can be used for the early detection of NPC. Studies of the corresponding proteins may have significances in tumor biology, novel drug development, and immunotherapy. However, analysis of more serum samples is needed to improve the statistical power and to validate this method as a clinically reliable assay. Efficient alternatives detected by ELISA could facilitate development of a blood test for NPC prediction. Testing a full panel of antibodies specific for NPC will be an important part of assay validation and eventually defines the clinical applicability.In summary, we identified immunogenic proteins and their autoantibodies in peripheral blood and generated a panel of antibody markers, which could have significant diagnostic, therapeutic and scientific values.