结直肠癌中CD8^(+)T细胞浸润特征及其预后意义

Characteristics of CD8^(+) T cell infiltration in colorectal cancer and their correlation with prognosis

目的CD8^(+)T细胞作为细胞毒性T细胞,可以通过释放穿孔素等方式直接杀伤肿瘤细胞,但其浸润水平与结直肠癌预后的关系,既往研究结果不尽相同。本研究旨在探讨CD8^(+)T细胞在结直肠癌肿瘤中心和浸润边缘的分布,并分析其与患者预后的关系。方法本研究采用回顾性队列研究方法,分析2009—2012年间,中山大学附属第六医院结直肠癌病理数据库中的221例结直肠癌患者临床病理资料。病例纳入标准:(1)经手术切除后病理证实为结直肠癌;(2)有完整随访信息。排除标准:(1)多原发癌;(2)合并炎性肠病、林奇综合征或家族性腺瘤性息肉病;(3)无石蜡切片;(4)术前接受过放疗或化疗。采用免疫组织化学染色方法计数患者石蜡切片中肿瘤中心及浸润边缘的CD8^(+)T细胞。同时选取22例新鲜冰冻结直肠癌组织标本,检测CD8B基因相对表达量,并与CD8^(+)T细胞计数进行相关分析。通过最小P值法选取肿瘤中心和浸润边缘CD8^(+)T细胞计数的最佳截断值,将患者分为高CD8^(+)T细胞浸润和低CD8^(+)T细胞浸润两组,采用Log-rank检验比较两组患者的总体生存,并采用Cox回归分析校正CD8^(+)T细胞浸润的预后意义。结果全组患者中男性118例,女性103例。在221例患者的切片中,肿瘤浸润边缘比肿瘤中心有更多的CD8^(+)T细胞浸润[M(范围):37(0~141)个/视野比14(0~106)个/视野,Z=-11.985,P<0.001],两者呈正相关趋势,差异有统计学意义(r=0.610,P<0.001)。肿瘤中心CD8^(+)T细胞计数与CD8B基因相对表达呈正相关趋势(r=0.524,P=0.012)。肿瘤中心和浸润边缘CD8^(+)T细胞数量的最佳截断值分别为2.6个/视野和24.8个/视野。术后中位随访83(1~117)个月,无论是肿瘤中心还是浸润边缘,高CD8^(+)T细胞浸润组患者的总体生存均优于低CD8^(+)T细胞浸润组(中位总体生存时间:84.1个月比73.5个月,P<0.001;84.2个月比75.9个月,P=0.002)。Cox回归分析证实,肿瘤中心高CD8^(+)T细胞浸润是总体生存的独立保护因素(HR=0.369,95%CI:0.168~0.812,P=0.013)。结论结直肠癌中CD8^(+)T细胞在肿瘤中心的浸润水平低于浸润边缘,且两者正相关。肿瘤中心CD8^(+)T细胞浸润水平与总体生存有关,可作为结直肠癌潜在的预后标志物。

Objective As cytotoxic T cells,CD8^(+)T lymphocytes can kill tumor cells by releasing perforin and other effector molecules,but the correlation between their infiltration level and the prognosis of colorectal cancer varies in previous studies.This study aims to explore the distribution of CD8^(+)T cells in tumor center and invasive margin of colorectal cancer,and to analyze their correlation with the prognosis of patients.Methods A retrospective cohort study was used to analyze the clinicopathological features of 221 patients with colorectal cancer from the colorectal cancer pathological database of the Sixth Affiliated Hospital of Sun Yat-sen University between 2009 and 2012.Case inclusion criteria:(1)colorectal cancers confirmed by postoperative pathology;(2)patients with follow-up data.Exclusion criteria:(1)multiple primary cancers;(2)inflammatory bowel disease,Lynch syndrome or familial adenomatous polyposis;(3)no available paraffin slides;(4)patients receiving preoperative radiotherapy or chemotherapy.A total of 221 patients met the criteria.Immunohistochemical staining was used to count the CD8^(+)T cells in tumor center and invasive margin in the paraffin slides.Meanwhile the relative expression of CD8B gene in 22 fresh freeze samples of colorectal cancer was detected.Then the correlation of the expression with CD8^(+)T cell density was examined.The patients were divided into high and low infiltration groups according to the level of CD8^(+)T cells.Log-rank test was applied to compare the overall survival of the two groups of patients,and Cox regression analysis was used to adjust the prognostic significance of CD8^(+)T cell infiltration.Results There were 118 males and 103 females.In 221 slides,CD8^(+)T cells infiltrating in invasive margin were more than those in tumor center[median(range):37(0-141)/field vs.14(0-106)/field,Z=-11.985,P<0.001],and the number of CD8^(+)T cell in the tumor center was positively correlated with those in invasive margin(r=0.610,P<0.001).The number of CD8^(+)T cell in tumor center was positively correlated with the relative expression of CD8B gene(r=0.524,P=0.012).Survival analysis showed that the overall survival of the high infiltration group was better than that of the low infiltration group both in tumor center and invasive margin(median survival:84.1 months vs.73.5 months,P<0.001;84.2 months vs.75.9 months,P=0.002).Cox regression analysis revealed that high CD8^(+)T cell infiltration in tumor center was an independent protective factor of overall survival(HR=0.369,95%CI:0.168-0.812,P=0.013).Conclusions The infiltration level of CD8^(+)T cells in tumor center is lower than that in invasive margin,and they are positively correlated.The level of CD8^(+)T cell infiltration in tumor center is related to overall survival and can be used as a potential pronostic marker.