建立鼻咽癌外泌体PLA-RPA检测新技术及在EBV抗体阳性人群中的筛查应用

EBV antibody testing has been used widely for nasopharyngeal carcinoma (NPC) screening. However, the previous epidemiologic studies indicated that the majority of the populations with serum EBV antibody positive are not associated with NPC. Previous studies on EBV infection associated biomarkers to increase the PPV of NPC is limited. To find more specific and suitable biomarkers for NPC screening, we performed quantitative proteomic study on exosomes from the two groups between NPC and nomal controls with positive EBV antibodies to identify new tumor markers for NPC screening and found that four membrane proteins sepcally expreesed in exosomes from NPC. We further established a new technology, termed as PLA-RPA-TMA,with a utra-sensitivity, to detect these markers on exosome, which turn out to be a good marker to different NPC patients at early stage from normal people with positive EBV antibodies. In this project, we will establish a novel PLA-RPA technology, which detect these four markers simulataneously. This new technology could be more simple and cost-effective as well as higher specificity than the old one and thus be more suitable to large-scale screening. We will identify its application value of screening NPC from both testing group and validted group in high risk areas. In addition, we will explore the roles of the exosome with the four positive markers in tumor development. EBV antibody screening combined with the second testing by PLA-RPA detection of NPC exosome would be more accurately to find out the persons with a high risk for NPC from a large people with positive EBV antibodies. This screening strategy would be helpul to improve the accuracy of EBV antibody test, ease the mental burden of most people with EBV antibody positive, and thus provide a theoretical basis for personalized prevention of NPC.

EBV抗体检测是鼻咽癌筛查最常用的手段，但在鼻咽癌高发区筛查发现EBV抗体阳性人群绝大多数并没有患鼻咽癌的风险。目前仅利用EBV感染等指标来提高鼻咽癌阳性预测值的研究效果均不理想。本项目前期利用定量蛋白质组技术从血浆外泌体中筛选出4个差异上调表达的膜蛋白，并创新地建立了一种高灵敏的PLA-RPA-TMA技术检测单个外泌体特异标志物，可用于区分早期鼻咽癌与EBV 抗体阳性正常人。本项目拟改进技术，建立PLA-RPA技术一管同步高灵敏检测这四个外泌体标志物，通过简化步骤，提高特异性，降低成本，使之适合大规模筛查应用；并拟在8年追踪的筛查标本库和广东鼻咽癌协助组标本库中验证其在筛查中应用价值，及初步探讨这几种外泌体的功能。我们利用EBV抗体初筛，外泌体标志物二次筛选的两步策略，将有助于精确筛选出鼻咽癌高危人群，从而减轻大部分抗体阳性人群不必要的精神负担，并为个性化鼻咽癌预防提供依据。

鼻咽癌患者血清特异性外泌体膜蛋白标志物含量低，传统方法难以检测。本研究建立了一种新的Aptamer-PCR-CRISPR/Cas12a技术，可直接检测100μl血清样本中EVs痕量膜蛋白（CD109和EGFR）。改进技术，用等温HCR（杂交链式反应）扩增技术替代PCR，建立了一种新的apta-HCR-CRISPR/Cas12a技术检测鼻咽癌血清EV标志物，此技术灵敏度与Aptamer-PCR-CRISPR/Cas12a技术相当，但不需要特殊仪器，操作简便，成本低； 采用此技术检测300例NPC患者和500例正常人血清nucleolin+ EV、CD109+ EV和EGFR+ EV水平，鼻咽癌患者血清nucleolin+ EV、CD109+EV和EGFR+ EV水平分别明显高于正常人；当血清nucleolin+ EV、CD109+ EV和EGFR+ EV诊断NPC的特异性为90%时，其检测灵敏度分别为78%、38%和71%；AUC分别为0.93、0.71和0.88。联合三者检测，诊断NPC特异性为90%时，检测灵敏度为96%，AUC为0.95。血清nucleolin+ EV、CD109+ EV和EGFR+ EV可作为诊断NPC的潜在血清标志物。采用此技术检测血清PD-L1+ EV水平的动态变化可作为NPC患者免疫治疗潜在的预测指标。另外， 基于PER串联信号放大技术，我们建立了高灵敏定量检测血清微量蛋白的iPTC技术，该技术的检测血清IL-15的下限相比于传统方法ELISA提高了153倍；采用iPTC技术血清IL-15可以作为NPC免疫治疗疗效预测的生物标志物（AUC = 0.8822, 95% CI 0.8083-0.9561, P <0.0001）。