弥漫大B细胞淋巴瘤中NF-κB/p65、PD-1、PD-L1表达相关性研究及其蛋白表达的临床意义

The correlation of NF-κB/p65, PD-1 and PD-L1 expressions and their clinical significance in diffuse large B-cell lymphoma

背景与目的:弥漫大B细胞淋巴瘤(diffuse large B-cell lymphoma,DLBCL)中NF-κB通路激活为重要发病机制,PD-1/PD-L1通路活化也与其相关,且NF-κB/p65、程序性死亡[蛋白]-1(programmed death-1,PD-1)、程序性死亡[蛋白]配体-1(programmed death ligand-1,PD-L1)蛋白表达与患者不良预后有关,但目前尚未见研究探讨它们之间的关系。探讨p65蛋白高表达与PD-1、PD-L1蛋白和mRNA的相关性,分析p65、PD-1、PD-L1蛋白表达与临床病理学特征、总生存期(overall survival,OS)的关系。方法:回顾性收集贵州医科大学附属医院病理科2010年1月-2017年12月90例DLBCL组织蜡块,采用免疫组织化学染色检测p65蛋白并分为p65蛋白高表达组和低表达组;免疫组织化学染色法检测各组PD-1蛋白;免疫组织化学双标记染色法检测各组肿瘤细胞PD-L1或肿瘤微环境细胞PD-L1(PD-L1 of tumor microenvironment cells,mP D-L1),即肿瘤浸润淋巴细胞(tumor-infiltrating lymphocyte,TIL)的细胞膜上表达的PD-L1蛋白;采用实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)检测各组中PD-1、PD-L1的mR NA表达水平;收集临床病理学资料并随访;对实验数据进行统计学分析。结果:90例样本中,p65高表达率为61.11%(55/90),PD-1阳性率为32.22%(29/90),肿瘤细胞PD-L1阳性率为24.44%(22/90),mPD-L1阳性率为28.89%(26/90);p65高表达与PD-1蛋白及mR NA均无相关性(P值均>0.05),但p65高表达与肿瘤细胞PD-L1或mP D-L1蛋白表达存在相关性(P=0.022,P=0.015),且在不同p65蛋白表达组间PD-L1 mRNA均值差异有统计学意义,p65高表达组相对较高(P=0.012);随访数据显示PD-1阳性与高国际预后指数(international prognostic index,IPI)评分有关(P=0.044),PD-L1阳性与高IPI评分及B症状的出现有关(P=0.007,P=0.001);Kaplan-Meier显示DLBCL中p65、PD-1、PD-L1、mPD-L1蛋白表达与患者OS相关,且p65高表达、PD-1阳性、PD-L1阳性、mPD-L1阳性患者OS相对较短(P值分别为0.038、0.015、0.028、0.010)。结论:DLBCL中PD-L1蛋白和mRNA上调表达与p65蛋白高表达相关;p65、PD-1、PD-L1、mPD-L1蛋白水平与患者OS较短有关,这些蛋白对临床及生存预后评估具有潜在价值。

Background and purpose: Activation of NF-κB pathway is an important mechanism in the pathogenesis of diffuse large B-cell lymphoma(DLBCL), which is also associated with the activation of programmed death-1(PD-1)/programmed death ligand-1(PD-L1) pathway, and protein expressions of NF-κB/p65, PD-1 and PD-L1 are associated with poor prognosis in patients,but the relationship between them has not been investigated. This study attempted to investigate the correlation between high p65 protein expression and protein and mRNA levels of PD-1 and PD-L1, and to analyze the correlation among p65, PD-1 and PD-L1 protein expressions and the clinicopathological characteristics and overall survival(OS). Methods: A total of 90 cases of DLBCL tissue wax blocks from Department of Pathology in Guizhou Medical University were enrolled in this study from Jan. 2010 to Dec. 2017. The p65 protein was detected by immunohistochemical staining, and DLBCL tissues were divided into high expression group and low expression group. PD-1 protein in each group was detected by immunohistochemical staining, while the expression of PD-L1 in tumor cells and tumor microenvironment cells(mPD-L1) [tumor-infiltrating lymphocyte(TIL)] was detected by immunohistochemical double labeling staining. The relative mRNA expressions of PD-1 and PD-L1 in the p65 high expression group and p65 low expression group were detected by real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR). The clinicopathological data were collected, and the follow-up was done. Finally, the experimental data were statistically analyzed. Results: Among all the samples, p65 positive rate was 61.11%(55/90), PD-1 positive rate was 32.22%(29/90), PD-L1 positive rate in tumor cells was 24.44%(22/90), and PD-L1 positive rate in tumor microenvironment cells(mPD-L1) was 28.89%(26/90). p65 high expression was not correlated with PD-1 protein or mRNA(P>0.05), whereas p65 high expression was correlated with PDL1 protein expression in tumor cells or microenvironment cells(P=0.022, P=0.015). There was a statistically significant difference in PD-L1 mRNA expression between groups, and the p65+ group was relatively higher(P=0.012). Clinical data showed that PD-1 positive rate was associated with high IPI score(P=0.044), and PD-L1 positive rate was associated with high IPI score and the occurrence of symptoms B(P=0.007, P=0.001). Kaplan-Meier analysis suggested that the expressions of p65, PD-1, PD-L1 and mPD-L1 were correlated with the OS of the patients with DLBCL, and the OS of patients who had p65 high expression, positive PD-1, PD-L1 and mPD-L1 was relatively short(P=0.038, 0.015, 0.028, 0.010). Conclusion: The up-regulated expressions of PD-L1 protein and mRNA are correlated with the high expression of p65 protein in DLBCL. Protein expressions of p65, PD-1, PD-L1 and mP D-L1 are related to shorter OS in patients, and these protein expressions have potential value for clinical and prognostic evaluation of survival.