从血浆exosomes中筛选和鉴定预测EBV抗体阳性的体检人群患鼻咽癌风险的指标

EBV antibody testing have been used widely for nasopharyngeal carcinoma (NPC) screening.Previously, our study showed that 100% of the patients with NPC can be screened out by EBV antibody testing. However, the further epidemiologic studies indicated that the vast majority of the populations of serum EBV antibody positive are without NPC since a very low positive predictive value (PPV:1.3%) was observed. Previous biomarker studies on EBV infection for increasing the PPV of NPC have not been worked out ideal. Tumor cells release exosomes into blood, consisting of an array of specific macromolecules derived from the originating cells, including proteins, lipids, and miRNA. Therefore, some specific biomarkers (e.g. proteins and miRNAs) for NPC should exist in the exosomes. To find more specific and suitable biomarkers for NPC screening, the plasma exosomes of the individuals of EBV antibody positive were isolated. These individuals were divided into two groups named NPC patients and normal without NPC respectively. Then we will perform proteomic study and miRNA profiling analyses on the plasma exosomes of both groups to identify new tumor markers for NPC and establish an efficient model for predicting the risk of NPC combined with EBV antibody testing. This model will improve the PPV of NPC, ease the mental burden of most people with serum EBV antibody positive, and providing a theoretical basis for personalized prevention of NPC.

EBV抗体已广泛应用于鼻咽癌筛查。我们先前大规模人群鼻咽癌筛查的研究已证实几乎100%的患者可通过EBV抗体筛选出来，但是追踪研究发现绝大多数EBV抗体阳性人群并不患鼻咽癌，其阳性预测值很低（1.3%）。目前仅从EBV感染出发来提高阳性预测值的研究其效果均不理想。由于肿瘤细胞可以产生外泌小体（exosome，Exo），含有特异的蛋白和miRNA标志物，并分泌入血易于检测。本项目前期从EBV抗体阳性体检人群中早期鼻咽癌患者和非鼻咽癌患者这两组血浆Exo中发现16种差异miRNA。本项目拟进一步扩大两组样本量，利用蛋白质组学和miRNA芯片技术，筛选和鉴定出鼻咽癌特异标志物，联合EBV抗体，建立鼻咽癌风险预测模型。该模型依据鼻咽癌细胞产生的特异标志物建立，将有助于提高EBV抗体阳性的体检人员鼻咽癌阳性预测值，减轻大部分人不必要的精神负担，并为个性化鼻咽癌预防提供理论基础。

采用定量蛋白组技术从血浆外泌体中筛选出10个NPC潜在诊断标志物，并建立了一种新的高灵敏、高特异的PLA-RPA-TMA技术检测血浆外泌体LMP1和 EGFR水平。血浆外泌体LMP1和EGFR标志物，区分NPC患者和VCA-IgA+/ EBNA1-IgA+正常人群的准确度AUC为0.906。 通过细胞因子芯片筛选出细胞因子MIF和CCL3是NPC诊断潜在标志物。血浆MIF、CCL3联合EBV抗体检测区分NPC患者和VCA-IgA+/ EBNA1-IgA+正常人群的准确性AUC为0.87。血浆CCL3高水平预示NPC患者预后不良，且高表达CCL3可促进NPC细胞增殖和迁移，抵抗化疗药物和放疗引起的细胞凋亡。 六种EBV抗体联合检测（VCAgp125 IgA, EBNA-1 IgA, EA-D IgA, EBNA-1 IgG, EAD IgG, 和 VCAp19 IgG），区别VCA-IgA+/EBNA1-IgA+正常人群与NPC患者的AUC为0.73，比单独一个EBV抗体指标诊断效果好。 利用芯片筛选的四个血浆外泌体miRNA标志物区分NPC患者和VCA-IgA+/ EBNA1-IgA+正常人群的准确度AUC为0.77。总之，血浆外泌体LMP1和EGFR标志物是区分NPC患者和VCA-IgA+/ EBNA1-IgA+正常人群的较好标志物。