| The strong association between the development of nasopharyngeal carcinoma (NPC) and Epstein-Barr virus (EBV) infection has aroused general concern. Actually, EBV-DNA could be detected in almost all the tissues of undifferentiated NPC, and the DNA is clonal, arising from a single EBV-infected cell. This suggests a role EBV plays in the course of malignant transformation. EBV infection has even successfully initiated carcinogenesis of the human fetal nasopharyngeal mucosa transplanted to nude mice.Elevated titers of the antibodies against EBV correlated antigens are frequently observed in serum of NPC patients. In addition to the well-known VCA-lgA (viral capsid antigen-immunoglobin A), there are several other antibodies against EBV correlated antigens such as EBNA ( EB nuclear antigen). In China, VCA-lgA is usually used as an indicator for screening of NPC However, It has never been shown for VCA-lgA titers to be correlated with theTNM classification, the clinical progress and the response to therapy of NPC. Therefore, VCA-IgA titers are believed to be able to indicate the risk of occurrence of NPC rather than to monitor the progress of the disease.It has been found in recent five years that the DNA deriving from a variety of malignant tumors may appear in peripheral circulation of patients. This enables us to monitor the advance of these malignant tumors by a nomnvasive approach. Lo et al were the first who could determinate EBV-DNA copies in plasma of 96% of NPC patients by FQ-PCR (fluorescence quantitative PCR) and established an excellent system of real-time quantitative determination of tumor-associated EBV-DNA in plasma of the patients with NPC.In the present study, the levels of EBV-DNA copies in plasma of pre- and post-treated NPC patients were examined, using FQ-PCR. Additionally, EB virus encoded RNA-1 (EBER-1) in primary tissues of NPC was detected with technique of non-isotopic in situ hybridization (NISH). Thus we try to describe the source of the plasma cell-free EBV-DNA of NPC patients and elucidate the clinical significance of quantitative analysis of plasma EBV-DNA of NPC patients.Materials and methods1. Materials66 plasma samples were obtained from 55 patients who were histologically confirmed as NPC and referred to the Department of Otorhinolaryngology or Radiation Oncology in the Second Affiliated Hospital of Zhejiang University between May 2000 and May 2001. The collected plasma samples consisted of 42 pre-treated and 24 post-treated samples. Among them, 11 patients offered double8samples of pre- and post- treated plasma. As normal controls, 30 plasma samples of blood donors were collected. Peripheral blood (5ml) was collected from each subject into an EDTA tube for isolation of plasma. 1.5 ml isolated plasma were taken and stored at -20癈 until further processing. 16 paraffin-embedded biopsy specimens taken lately from these NPC patients were selected for NISH experiment.Among 55 NPC patients, 40 cases were males and 15 cases were females, aging from 19 to 75 (median ages 53.0). All the patients were evaluated by nasopharyngeal endoscopy and computed tomography, and classified according to the U1CC/AJCC staging criteria (1997). There were 5 cases classified as stage I, 25 as stage II, 10 as stage UL, and 15 as stage IV. 30 control donors were composed of 22 males and 8 females, aging from 34 to 50 (average ages 43.0)2. Methods2 1 Quantitative determination of plasma EBV-DNA1.5ml plasma samples were centrifuged at 12000 rpm for 10 minutes at 4'C. The deposits were collected and mixed with the DNA-extracting agent. Mixture was heated in a boiling water bath for lOminutes, quickly chilled in an ice/water bath, and then centrifuged at 10000 rpm for 5minutes at 4'C. 2 u 1 of supernatant was amplified. The sequences of the primers were 5' -TCTCTGCCTCCAGGC AAG-3 ' and 5' -AGAGGGCCTGTCCACCGT-3 ', and the sequence of dual-labeled fluorescent probe was 5' -(FAM)CTGTCTGTAAAGTCCAGCCTCC-(TAMRA) -3'. The amplification procedure was programmed as follows: (D pre-d...
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