进展期胃癌中EB病毒感染率及EB病毒参与免疫调节的机制探索

Exploration of Epstein-Barr virus infection rate in advanced gastric cancer and the potential mechanism of Epstein-Barr virus involvement in immune regulation

目的分析进展期胃癌中EBV阳性率及EBV参与机体免疫调节的可能机制。方法 99例进展期胃癌患者入选该研究,收集患者化疗前肿瘤组织标本99例和化疗后肿瘤组织标本108例,以及相应61例人源化胃癌移植瘤(patient-derived xenograft,PDX)组织,用显色原位杂交方法检测所有组织中EBV-RNA(EBER)状态;荧光定量PCR法检测患者血浆及淋巴细胞中EBV-DNA拷贝数;在胃癌细胞AGS中导入EBV相关BZLF1蛋白构建EBV阳性细胞,RNA测序方法分析EBV阴性及阳性胃癌细胞中差异表达的分子或通路,尤其是跟免疫相关的通路。结果 99例进展期胃癌患者化疗前标本中EBV阳性率为1.01%(1/99),且EBV状态不受化疗影响,PDX组织中EBV阳性率高达21.31%(13/61)。在8例原发肿瘤组织EBV阴性而相应PDX组织EBV阳性的患者中,5例(62.50%)患者的外周血存在EBV潜伏感染。胃癌细胞AGS中导入BZLF1蛋白后,与其配对的亲本细胞相比,筛选出差异表达分子537个,其中上调表达分子265个,下调表达分子272个,通路富集分析表明,上调通路有Jak-STAT通路、PPAR通路、细胞因子-细胞因子受体相互作用通路等,这几条通路均与免疫调节相关。结论我国进展期胃癌中EBVaGC比例较低,PDX组织中EBV阳性率较高可能是由于小鼠免疫缺陷促进潜伏感染的EBV激活复制,EBV感染可能通过激活机体固有免疫而从免疫治疗中获益,确切机制有待深入探索。

Objective Aimed to analyze the positive rate of EBV in advanced gastric cancer and the possible mechanisms of EBV involvement in immune regulation. Method Biopsy tumor tissues before(n = 99) and after(n = 108) treatment and 61 PDX(patientderived xenograft) tissues from 99 advanced gastric cancer patients(AGC) were detected for EBV-RNA(EBER) status by Chromogenic in situ hybridization(CISH). Fluorescent quantitative PCR was used to detect EBV-DNA copy number in plasma and peripheral blood lymphocytes of patients. Constructing EBV positive cell by transfecting EBV-BZLF1 protein into AGS cell, RNAseq was conducted to analyze the differentially expressed molecules or pathways, especially immune-related pathways in paired EBV negative and positive AGS cells. Result EBV positive rate in AGC patients was only 1.01%(1/99) in our cohort and EBV status wasn’t changed by treatment,however, the EBV positive rate was 21.31%(13/61) in PDX tissues. Among 8 patients with EBV negative in primary tumors and positive in corresponding PDX tissues, 5 patients(62.50%) were detected to carry EBV latent infection in plasma or lymphocytes. Compared with the paired parental cells, AGS cells with transfected BZLF1 protein had 537 differentially expressed molecules, of which 265 molecules were up-regulated and 272 molecules were down-regulated. Pathway enrichment analysis indicated that Jak-STAT pathway,PPAR pathway, and Cytokine-cytokine receptor interaction pathway were up-regulated in EBV positive cells, which were related to immune regulation. Conclusion The proportion of EBVaGC was very low in Chinese AGC patients and the higher proportion in PDX tissues might be due to immune deficiency in mice that promoted latent EBV reactivation and replication. EBVaGC benefited from immunotherapy possibly by activating the innate immunity, the exact mechanism needs to be explored in depth.