【作者】 乔治；

【导师】 李荣； 徐迎新；

【作者基本信息】 中国人民解放军军医进修学院 ， 普通外科， 2009， 博士

【摘要】 研究背景:胃癌是我国最常见的消化道恶性肿瘤,尽管手术、化疗、新辅助化疗、放疗等治疗方法不断发展,但进展期胃癌患者预后仍然欠佳,因此迫切需要开展胃癌综合治疗新策略研究。近年来生物技术的迅猛发展为此奠定了基础。目前已知过继性细胞免疫治疗（adoptive cellular immunotherapy,ACI）对免疫原性较强的实体肿瘤如黑色素瘤和肾癌等治疗效果良好,但其对胃癌等弱免疫原性肿瘤疗效尚不明确。根据肿瘤免疫编辑学说,胃癌患者机体预存免疫状态与肿瘤发病、进展、治疗及预后均密切相关。因此建立胃癌患者免疫状态检测技术平台并评估其预存免疫状态,从而将免疫指标加入胃癌疗效评价体系,对确定其术后免疫治疗方案及预后评价均有着重要的参考价值。胃癌术后早期患者创伤反应刚过,机体免疫功能受到创伤进一步打击,且身体虚弱,伤口有待愈合,尚不适合进行全身化疗。而此时体内肿瘤负荷则减到最低,应该是进行免疫细胞过继转移的最佳时机。术后早期将在体外培养的多种免疫活性细胞如自体肿瘤抗原致敏的树突状细胞（DC）、细胞因子诱导的杀伤细胞（CIK）和肿瘤特异性细胞毒T淋巴细胞（CTL）回输体内,可能拮抗体内免疫抑制因素,调动并提高机体抗肿瘤免疫功能,从而取得更为满意的疗效。因此,术后早期开展多种免疫活性细胞联合过继转移治疗具有重要意义。研究目的:建立免疫状态检测技术平台以检测胃癌患者预存免疫状态并作为疗效评价指标,构建胃癌患者手术联合免疫活性细胞过继转移治疗数据库及疗效评价体系,观察术后早期多种免疫活性细胞联合过继转移治疗对患者预后的影响,为制定新的胃癌综合治疗方案奠定基础。研究方法: 1.建立流式细胞术检测包括外周血树突状细胞（DC）、调节性T细胞（Treg）、记忆性T细胞、自然杀伤细胞（NK）、自然杀伤T细胞（NKT）、辅助性T淋巴细胞（Th1/Th2）、细胞毒性T淋巴细胞（Tc1/Tc2）以及代表免疫细胞功能的细胞内细胞因子（IFN-γ及IL-4）在内的胃癌患者免疫状态检测技术平台;2.检测25例解放军总医院普通外科2008年10月21日至2008年12月5日新入院术前胃癌患者及25例正常健康献血者外周血样本,对胃癌患者预存免疫状态进行初步研究;3.建立胃癌患者综合治疗数据库及疗效评价平台,并对综合治疗后胃癌患者随访,开展术后早期多种免疫活性细胞联合过继转移治疗回顾性研究。研究结果:1.胃癌患者预存免疫状态检测结果示:胃癌患者外周血调节性T细胞较正常对照组显著增多;胃癌患者外周血Th1及Tc1亚群较正常对照组明显减少,并出现Th1/Th2及Tc1/Tc2漂移呈Th2及Tc2优势现象;胃癌患者外周血记忆性T细胞CD4⁺T_CM、CD4⁺T_EM、CD8⁺T_CM亚群均较正常对照组明显升高,但是在外周主要承担杀伤性效应的CD8⁺T_EM亚群不但不升高,反而较正常人群略有下降;胃癌患者外周血DC1、DC2及DC1/DC2较正常对照组无明显改变;胃癌患者外周血NK及NKT细胞较正常对照组无明显改变;2.共有效随访68例综合治疗后胃癌患者,COX比例风险模型多因素分析显示:患者年龄、手术类型、临床分期及术后ACI治疗为影响胃癌患者预后的独立因素（P<0.05）。单因素Kaplan-Meier模型分析及Log-Rank检验结果显示患者年龄、手术类型、临床分期及术后早期ACI治疗（手术后14天内）是影响胃癌患者术后预后的相关因素（P<0.05）,而患者性别、病理类型、术后化疗、术后ACI治疗次数对患者预后没有显著性影响（P>0.05）。研究结论:1.流式细胞术检测胃癌患者免疫状态技术平台方法稳定,临床应用性良好;2.胃癌患者预存免疫状态较正常人明显低下,评估患者预存免疫状态并将免疫指标加入胃癌疗效评价体系对确定其治疗方案及疗效评价有着重要意义；3.患者年龄、手术类型、临床分期及术后ACI治疗为影响胃癌患者预后的独立因素,较晚的发病年龄（>50岁）、施行根治性手术、较早的临床分期及术后早期ACI治疗（手术后14天内）可延长术后胃癌患者生存期,而患者性别、病理类型、术后化疗、术后ACI治疗次数对患者预后没有显著性影响。本项研究表明,术后早期免疫活性细胞过继转移治疗可延长胃癌患者手术后生存期,为提高胃癌患者综合治疗水平开创了新的前景。更多还原

【Abstract】 Background: Gastric cancer is the most common malignant tumor of digestive tract in China. Despite of the development of various treatments such as surgery, chemotherapy, neoadjuvant chemotherapy and radiotherapy, the prognosis of gastric cancer patients at advanced stage still remains poor. The possibility of applying immunotherapy for gastric cancer would therefore be highly desirable. Adoptive cellular immunotherapy （ACI） have therapeutic activity in with high immunogenic tumor such as melanoma and kidney cancer, however, it’s still unclear in gastric cancer.According to tumor immunoediting hypothesis, pre-existing immunity of gastric cancer patients is closely related to onset and progress of tumor and the poor prognosis. Therefore, establishing methods to assess pre-existing immunity and adding immunological parameters into the prognostic evaluation system of gastric cancer are significant to evaluate postoperative therapeutic strategy and efficacy.The immunological function of early postoperative patients is decreased significantly, and it is not suitable for applying systemic chemotherapy at this point. However, the tumor load in vivo is reduced to minimum. Therefore, it is suggested that early postoperative infusion of multiple immunologicly competent cells, such as DC, CIK and CTL, should improve immune function of patients and achieve more satisfactory results.Objective: This study was designed to establish methods for assessing pre-existing immunity of gastric cancer patients, establish database and prognostic evaluation system of gastric cancer patients after comprehensive treatment, and observe effect of early postoperative ACI on prognosis of gastric cancer patients.Methods: 1. The flow cytometric immune function assessing system was established, including detection of peripheral DC, Treg, memory T cells, NK, NKT, Th1/Th2, Tc1/Tc2 cells and intracellular cytokine （IFN-γand IL-4） by four-color flow cytometry; 2. Preliminary analysis of pre-existing immunity of preoperative gastric cancer patients （n=25） and healthy controls （n=25） was performed by detecting subsets of DC1 （Lin^-DR⁺CDl11c⁺CD123^-）, DC2 （Lin^-DR⁺CD11c^-CD123⁺）, NK （CD3^-CD56⁺）, NKT （CD3⁺ CD56⁺）, Treg （CD4⁺CD25⁺FOXP3⁺）, CD4⁺T_CM （CD4⁺CD45RO⁺CCR7⁺CD45RA^-）, CD4⁺T_EM （CD4⁺CD45RO⁺CCR7^-CD45RA^-）, CD8⁺T_CM （CD8⁺CD45RO⁺CCR7⁺CD45RA^-）, CD8⁺T_EM （CD8⁺CD45RO⁺CCR7^-CD45RA^-）, Th1 （CD4⁺IFN-γ⁺）, Th2 （CD4⁺IL-4⁺）, Tc1 （CD8⁺IFN-γ⁺） and Tc2 （CD8⁺IL-4⁺） cells in peripheral blood; 3. A retrospective database of 93 gastric cancer patients following surgical treatment combined with chemotherapy and/or immunotherapy from December 2004 to March 2008 was constructed firstly. Data were censored at time of death, loss to follow-up or January 1st, 2009. Survival was estimated using the Kaplan-Meier method. The factors affecting the survival time were evaluated by Cox proportional hazard model and Log-Rank test.Results: 1. The results of preoperative gastric cancer patients show: the CD4⁺CD25⁺FOXP3⁺ Treg of gastric cancer patients were significantly higher than the normal control group （1.993±0.830% Vs 1.229±0.656%, P <0.01）; the ratios of Th1/PBL, Tc1/PBL, Th1/Th2 and Tc1/Tc2 in gastric cancer patients were significantly lower than the normal control group （3.047±1.710 % Vs 6.242±4.078 % , 6.393±5.235 % Vs 14.171±8.984 %, 1.215±0.219 % Vs 2.552±2.343 %, 1.306±0.289 % Vs 17.200±25.930 %, P<0.05）; the CD4⁺T_CM, CD4⁺T_EM, CD8⁺T_cm of gastric cancer patients were significantly higher than the normal control group （4.585±2.502% Vs 2.765±1.942%, 18.682±7.374% Vs 13.032±4.059 %, 0.455±0.472% Vs 0.127±0.165%, P <0.05）, but there was no significant difference of CD8⁺T_EM between them （8.379±3.431% Vs 8.733±3.048%, P> 0.05）; there was no significant difference of DC1, DC2 and DC1/DC2 in peripheral blood between gastric cancer patients and normal control （0.201±0.095% Vs 0.198±0.087%, 0.081±0.032% Vs 0.093±0.046%, 2.693±1.636% Vs 2.561±1.486%, P> 0.05）; there was no significant difference of NK and NKT in peripheral blood between gastric cancer patients and normal control （19.209±14.116% Vs 20.306±8.844%, 8.481±5.378% Vs 6.156±3.934%, P> 0.05）. 2. Complete follow-up data were obtained from 68 cases of patients, majority of who were advanced stage ones. In total, 35 patients died, giving median survival time of 21 months. The estimated 3-year overall survival was 36%. In univariate analysis, age, clinical stage, type of surgery and early postoperative adoptive immunotherapy （less than 14 days after operation） were significant prognostic factors related to overall survival time （P <0.05）. Gender, histopathologically diffuse type, adjuvant chemotherapy and numbers of immunotherapy were found to have no effect on survival （P> 0.05）. In multivariate analysis, age, radical surgery, clinical stage, early postoperative adoptive immunotherapy （less than 14 days after operation） were found to be the statistically significant prognostic factors related to survival （P <0.05）.Conclusion: 1. The flow cytometric immune function assay was a stable method for assessing pre-existing immunity of gastric cancer patients; 2. The pre-existing immunity was poor in preoperative gastric cancer patients, which might be related to the onset and progress of tumor and the poor prognosis. Immunological parameters might be useful predictors to evaluate prognosis of gastric cancer patients; 3. The factors including age, clinical stage, radical surgery and early postoperative adoptive immunotherapy had significant effect on survival of postoperative gastric cancer patients. Early postoperative adoptive immunotherapy couid be a beneficial adjuvant therapy to improve clinical outcomes of gastric cancer patients.更多还原