免疫检查点B7同源物4在子宫内膜癌组织中的表达及其临床意义

Expression and significance of immune checkpoint B7-homolog 4 in endometrial cancer

目的探讨免疫检查点B7同源物4(B7-H4)在子宫内膜癌组织中的表达及其临床意义。方法选择2009年至2019年中国医学科学院北京协和医院收治的833例子宫内膜癌患者。采用免疫组化EnVision二步法检测子宫内膜癌组织中B7-H4、错配修复(MMR)、p53、细胞程序性死亡配体1(PD-L1)蛋白的表达及CD_(8)^(+)T淋巴细胞密度;采用第一代测序技术(Sanger法)对子宫内膜癌组织进行分子分型检测。比较不同临床病理特征、分子分型、PD-L1蛋白表达、CD_(8)^(+)T淋巴细胞密度的子宫内膜癌组织中B7-H4蛋白阳性表达率的差异;对其中随访时间≥3个月的664例患者进行预后[包括无复发生存率(RFS)和疾病特异性生存率(DSS)]分析,其中位随访时间为31个月(范围:4~121个月),采用Cox比例风险回归模型对影响子宫内膜癌患者预后的相关因素进行单因素和多因素分析。结果(1)833例子宫内膜癌患者的中位年龄为58岁(范围:25~88岁);病理类型:子宫内膜样癌595例,非子宫内膜样癌238例;手术病理分期:Ⅰ期542例,Ⅱ期38例,Ⅲ期173例,Ⅳ期45例,不详35例。590例患者完成了分子分型检测,其中POLE突变型50例,错配修复缺陷(MMR-d)型163例,无特异性分子改变(NSMP)型246例,p53突变型131例。(2)在子宫内膜癌的细胞膜和细胞质中B7-H4蛋白呈棕黄色阳性表达,子宫内膜癌组织中B7-H4蛋白的阳性表达率为71.5%(596/833)。不同年龄、手术病理分期、肿瘤级别、病理类型、肌层浸润深度、有无淋巴脉管间隙浸润、分子分型患者的B7-H4蛋白阳性表达率分别比较,差异均有统计学意义(P均<0.05);而不同PD-L1蛋白表达及CD_(8)^(+)T淋巴细胞密度患者的B7-H4蛋白阳性表达率分别比较,差异则均无统计学意义(P均>0.05)。B7-H4蛋白阳性患者的5年RFS和DSS(83.9%、87.3%)分别与B7-H4蛋白阴性患者(77.2%、78.1%)比较均有增高趋势,但分别比较,差异均无统计学意义(P=0.053,P=0.083)。(3)单因素分析显示,不同年龄、肿瘤级别、手术病理分期、病理类型、肌层浸润深度、淋巴脉管间隙浸润、分子分型患者的5年RFS和DSS分别比较,差异均有统计学意义(P均<0.01);不同CD_(8)^(+)T淋巴细胞密度及PD-L1蛋白表达患者的5年RFS(P=0.184,P=0.113)及5年DSS(P=0.549,P=0.247)分别比较,差异均无统计学意义。进一步对不同分子分型、CD_(8)^(+)T淋巴细胞密度、PD-L1蛋白表达的子宫内膜癌患者进行分析,结果显示,在分子分型为NSMP型、CD_(8)^(+)T淋巴细胞低密度、PD-L1蛋白阴性表达的子宫内膜癌患者中,除PD-L1蛋白阴性子宫内膜癌患者中的B7-H4蛋白阳性患者的5年DSS与B7-H4蛋白阴性患者比较无显著差异(P=0.060)外,其他情况下B7-H4蛋白阳性患者的5年RFS和DSS均显著高于B7-H4阴性患者(P均<0.05)。多因素分析显示,在NSMP型子宫内膜癌患者中,B7-H4蛋白阳性表达是影响5年RFS(HR=0.27,95%CI为0.09~0.78,P=0.016)和5年DSS(HR=0.16,95%CI为0.05~0.58,P=0.005)的独立因素;在CD_(8)^(+)T淋巴细胞低密度的子宫内膜癌患者中,B7-H4蛋白阳性表达是影响5年RFS的独立因素(HR=0.45,95%CI为0.26~0.80,P=0.006),但并不是影响5年DSS的独立因素;在PD-L1蛋白阴性表达的子宫内膜癌患者中,B7-H4蛋白表达并不是影响5年RFS的独立因素。结论子宫内膜癌组织中B7-H4蛋白高表达,且其高表达与具有良好的临床病理特征、分子分型为p53突变型和NSMP型、免疫表型为CD_(8)^(+)T淋巴细胞低密度患者的良好预后密切相关。

Objective To investigate the expression of B7 homolog 4(B7-H4)and its clinical significance in endometrial cancer.Methods A total of 833 patients with endometrial cancer admitted to Peking Union Medical College Hospital,Chinese Academy of Medical Sciences from 2009 to 2019,were enrolled.The expression of B7-H4,mismatch repair(MMR),p53,programmed cell death ligand 1(PD-L1)protein,and CD_(8)^(+)T lymphocyte density in endometrial cancer tissues were detected by the EnVision two-step method of immunohistochemical staining.First-generation sequencing(Sanger method)was used to determine molecular subtyping of endometrial cancer.Theχ2 test was used to compare the differences in positive expression rate of B7-H4 protein in endometrial cancer tissues with different clinicopathological features and molecular subtyping,PD-L1 protein expression,and CD_(8)^(+)T lymphocyte density.Survival analyses[including recurrence-free survival(RFS)and disease-specific survival(DSS)]were performed for 664 patients with follow-up time≥3 months,with a median follow-up time of 31 months(range:4-121 months),and the Cox proportional hazards regression model was used to analyze the relevant factors affecting the prognosis of patients with endometrial cancer.Results(1)The median age of 833 patients was 58 years(range:25-88 years);pathological type:595 with endometrioid carcinoma,238 with non-endometrioid carcinoma;surgical-pathological staging:542 cases at stageⅠ,38 cases at stageⅡ,173 cases at stageⅢ,and 45 cases at stageⅣ.Molecular subtyping was performed in 590 patients,including 50 with POLE mutation,163 with mismatch repair defect(MMR-d)type,246 with nospecific molecular change(NSMP)type,and 131 with p53 mutation subtype.(2)B7-H4 protein was expressed with brownish-yellow stainind in the cell membrane and cytoplasm of endometrial carcinoma,and the positivity rate of B7-H4 protein was 71.5%(596/833).The positivity rates of B7-H4 protein among patients with different age,surgical-pathological stage,tumor grade,pathological type,depth of muscular invasion,presence or absence of lymphovascular space invasion,and molecular subtype were significantly different(all P<0.05).The positivity rates of B7-H4 protein among patients with different PD-L1 protein expression and CD_(8)^(+)T lymphocyte density were not significantly different(P>0.05).The 5-year RFS(83.9%)and DSS(87.3%)of B7-H4 protein-positive patients had an increasing trend compared with the 5-year RFS(77.2%)and DSS(78.1%)of B7-H4 protein-negative patients,but there were not statistically significant differences(P=0.053,P=0.083).(3)Univariate analysis showed that the 5-year RFS and DSS of patients with different age,tumor grade,surgical-pathological stage,pathological type,depth of muscular invasion,lymphovascular space invasion,and molecular subtype were significantly different(all P<0.01).There were no significant differences in 5-year RFS(P=0.184,P=0.113)and DSS(P=0.549,P=0.247)among patients with different CD_(8)^(+)T lymphocyte density and PD-L1 protein expression.Further analysis according to molecular subtype,the results of CD_(8)^(+)T lymphocyte density and PD-L1 protein expression showed that the 5-year RFS and DSS of B7-H4 protein-positive patients were higher than those of B7-H4 protein-negative patients with NSMP subtype,low density of CD_(8)^(+)T lymphocyte and PD-L1 protein-negative endometrial carcinoma(all P<0.05),however,there was no significant difference in 5-year DSS between B7-H4 protein-positive patients and B7-H4 protein-negative patients with PD-L1 protein-negative endometrial cancer(P=0.060).Multivariate analysis showed that positive expression of B7-H4 protein was an independent factor for 5-year RFS(HR=0.27,95%CI:0.09-0.78,P=0.016)and DSS(HR=0.16,95%CI:0.05-0.58,P=0.005)in patients with NSMP subtype endometrial carcinoma.In patients with low-density CD_(8)^(+)T lymphocytes endometrial cancer,positive expression of B7-H4 protein was an independent factor for 5-year RFS(HR=0.45,95%CI:0.26-0.80,P=0.006),but it was not an independent factor for 5-year DSS.In patients with PD-L1 protein-negative endometrial cancer,B7-H4 protein was not an independent factor for 5-year RFS.Conclusion B7-H4 protein expressed highly in endometrial carcinoma tissues,and its high expression is closely related to clinicopathological features,molecular subtype of p53 mutant and NSMP,and the favorable prognosis of patients with low density of CD_(8)^(+)T lymphocyte immunophenotype endometrial carcinoma.