【作者】 刘方方；

【导师】 刘惠敏；

【作者基本信息】 第二军医大学 ， 病理学与病理生理学， 2011， 硕士

【摘要】 研究背景:弥漫性大B细胞淋巴瘤(diffuse large B-cell lymphoma, DLBCL)是一种最常见的侵袭性淋巴瘤,其形态学、免疫表型、遗传学特点及临床预后各异。DLBCL常见临床表现是快速增大的淋巴结,也可以原发于任何结外组织器官,结外最常见的部位是胃肠道。目前,原发性胃肠道弥漫性大B细胞淋巴瘤(primary gastrointestinal diffuse large B-cell lymphoma, PGI-DLBCL)资料非常有限,在临床上恶性程度及侵袭性比结内DLBCL高,且与消化道的一般疾病或消化道癌的表现难以区别。因此发现时分期较晚、误诊率较高。由于手术切除存在致死性的并发症,近年来将化疗作为胃肠道DLBCL的首选治疗,而部分患者对常规化疗不敏感,预后差。故进一步阐明PGI-DLBCL的发病机制,寻找合适的治疗靶点对提高PGI-DLBCL的疗效仍然是当务之急。核转录因子κB(nuclear factorκB, NF-κB)是一类蛋白质核转录因子,在多种肿瘤中存在着选择性激活或表达异常。目前国内外绝大多数学者认为,NF-κB持续活化是活化B细胞(activate B-cell like, ABC-DLBCL)预后不好的原因之一,而胱冬肽酶募集结构域(caspase recruitment domain 11, CARD11)是NF-κB信号通路上游的重要蛋白分子,丰富的临床前资料也证明了CARD11在DLBCL中的重要性。目前,国内外尚未发表有关在PGI-DLBCL中,CARD11与NF-κB的表达与免疫分型,临床特征和生物学行为的预后相关性研究报道。研究目的:检测原发性胃肠道弥漫性大B细胞淋巴瘤中CARD11,NF-κB/p65、NF-κB/p50,CD10,Bcl-6,Mum-1,Bcl-2,Ki-67的表达,并结合临床及随访资料,系统探讨CARD11与NF-κB的表达与免疫分型,临床特征和生物学行为的关系,并筛选PGI-DLBCL预后因素,为进一步寻找更加可靠的预测预后的分子标记物提供新的理论基础,并为临床联合应用靶向化疗药物提供新的理论依据。研究方法:选取1998年5月至2010年6月间长征医院原发性胃肠道弥漫性大B细胞淋巴瘤54例石蜡包埋组织样本,并收集相关科室和临床随访资料;采用免疫组织化学Envision法检测CD10、Bcl-6、Mum-1对其进行免疫分型,检测CARD11、NF-κB/p65、NF-κB/p50的表达情况;观察、评估各因子阳性表达率及表达强度,分析其相关性及与免疫分型、临床特征及凋亡指数Bcl-2、增殖指数Ki-67的关系,筛选预后相关因素。应用SPSS统计软件分析各因子表达相互间关系及与PGI-DLBCL生物学行为的关系;Kaplan-Meier生存分析及Log-Rank时序检验分析各相关因素与PGI-DLBCL预后的关系;Cox回归模型分析多因素预后分析。研究结果:1. (1)本组54例PGI-DLBCL中CD10阳性15例(28%),Bcl-6阳性37例(69%),Mum1阳性44例(81%) ,CARD11阳性表达率为70%(38/54), NF-κB/p65阳性表达率为72%(39/54),NF-κB/p50阳性表达率为65%(35/54)。(2)根据检测结果将PGI-DLBCL分为生发中心B细胞型(GCB型)19例(35%),ABC型35例(65%)。GCB亚型中CARD11、NF-κB/p65、NF-κB/p50的表达率分别为26.3%、25.6%、25.7%,ABC亚型中CARD11、NF-κB/p65、NF-κB/p50的表达率分别为73.7%、74.4%、74.3%,ABC亚型中CARD11、NF-κB/p65、NF-κB/p50的表达率均明显高于GCB亚型(P<0.05)。(3) CARD11在NF-κB/p65、NF-κB/p50低表达组(阴性和阳性)与高表达组(中度阳性和高度阳性)之间均有显著差异性(r=0.314, r=0.322, P<0.05),其中CARD11在NF-κB/p65、NF-κB/p50低表达组中的表达均显著低于其在NF-κB/p65、NF-κB/p50高表达组中的表达。2. (1) CARD11的表达程度与患者性别、年龄、发病部位、临床分期、浸润深度、淋巴结受累、结外侵犯部位、肿块大小、LDH、ECOG评分、IPI、B症状均无显著相关性(P>0.05)。NF-κB/p65的表达程度与结外侵犯部位有显著差异(P<0.05),其阳性表达率在结外侵犯部位各组间关系为:“≥2”组(76.9%)>“0~1”组(70.7%),而与患者性别、年龄、发病部位、临床分期、浸润深度、淋巴结受累、肿块大小、LDH、ECOG评分、IPI、B症状均无显著相关性(P>0.05)。NF-κB/p50的表达程度与临床分期、结外侵犯部位、LDH、IPI均有显著差异(P<0.05),其阳性表达率在临床分期各组间关系为:“Ⅲ/Ⅳ”组(66.7%)>“Ⅰ/Ⅱ”组(63.6%),在结外侵犯部位各组间关系为:“≥2”组(76.9%)>“0~1”组(61.0%),在LDH各组间关系为:“升高”组(75.0%)>“正常”组(60.5%),在IPI各组间关系为:低危组(60.6%)<低中危组=高中危组(66.7%)<高危组(77.8%),而与患者性别、年龄、发病部位、浸润深度、淋巴结受累、肿块大小、ECOG评分、B症状均无显著相关性(P>0.05)。(2) CARD11在Bcl-2及Ki-67低表达组和高表达组之间均无显著差异。NF-κB/p65、NF-κB/p50在Bcl-2低表达组与高表达组之间均有显著差异性(r=0.350, r=0.328, P<0.05)。NF-κB/p65在Ki-67低表达组与高表达组之间有显著差异性(r=0.271, P<0.05),而NF-κB/p50在Ki-67中无显著差异(P>0.05)。3.本组54例PGI-DLBCL患者性别、结外侵犯部位、LDH、IPI、免疫分型、NF-κB/p65皆与生存期有显著差异性(P<0.05),其中男性组平均生存期显著低于女性组;结外侵犯部位“≥2”组平均生存期显著低于“0~1”组;初诊时LDH升高生存率明显降低;随着危险因素的增多,IPI中高/高中危组平均生存期显著低于低/低中危组;免疫亚型中ABC型平均生存期显著低于GCB型;NF-κB/p65阳性表达患者平均生存期显著低于阳性表达患者。而年龄、发病部位、临床分期、浸润深度、淋巴结受累、肿块大小、ECOG评分、B症状、化疗方法,以及CARD11、NF-κB/p50表达水平各分组间平均生存期无显著差异(P>0.05)。结外侵犯部位和NF-κB/p65表达水平进入最终Cox预后评估模型,预后指数判断预后有较好的吻合率。结论:1.在PGI-DLBCL中,ABC亚型中CARD11、NF-κB/p65、NF-κB/p50的表达率均明显高于GCB亚型,且CARD11与NF-κB/p65、NF-κB/p50的表达程度均呈正相关。表明在ABC亚型中CARD11与NF-κB有协同作用,证明CARD11作为上游信号传导分子激活NF-κB,进而对PGI-DLBCL的发生发展具有一定作用,提示CARD11可以作为ABC-DLBCL有吸引力的新的治疗靶点。2.在PGI-DLBCL中,NF-κB/p65的表达程度与结外侵犯部位有显著差异,NF-κB/p50的表达程度与临床分期、结外侵犯部位、LDH、IPI均有显著差异,二者均与凋亡指数Bcl-2呈正相关,说明NF-κB抑制肿瘤细胞凋亡,并和PGI-DLBCL的发展和侵袭呈正相关。结合免疫分型,NF-κB可预测PGI-DLBCL的发展程度。3.在PGI-DLBCL中,结外侵犯部位、LDH、IPI、免疫分型、NF-κB/p65皆可作为评判PGI-DLBCL预后的因素。其中结外侵犯部位和NF-κB/p65表达水平是影响PGI-DLBCL预后的主要因素,预后指数可用于预后判断,高预后指数预示危险度增大,生存期缩短。更多还原

【Abstract】 Background:Diffuse large B-cell lymphoma(DLBCL) represents the most common type of aggressive lymphoma. DLBCL is heterogeneous with respect to morphology, immunophenotype, biology, clinical presentation and outcome. The common clinical manifestation of DLBCL is rapidly enlarging lymph nodes, and it can also be primary in any extranodal tissues and organs of which the most common site is extranodal gastrointestinal.Currently, the primary gastrointestinal diffuse large B cell lymphoma (PGI-DLBCL) data is very limited. The degree of malignancy and invasiveness is higher than nodal DLBCL in clinic, and it was difficult to distinguish from the gastrointestinal common disease and gastrointestinal cancer.Therefore, when found, PGI-DLBCL is at clinical later stage and hence misdiagnosis rate is higher. Because operation have many fatal complications, the chemotherapy treatment as the preferred choice of gastrointestinal DLBCL, but some patients are not sensitive to conventional chemotherapy and have poor prognosis. Further elucidating the pathogenesis of DLBCL and searching for potential therapeutic targets remain a priority to improve the poor prognosis.Constitutive activity of the NF-κB pathway may contribute to the poor prognosis of patients with activated B cell–like (ABC) subgroup of DLBCLs, CARD11 is the important upstream signal protein in the signaling pathway of NF-κB. Furthermore, various pre-clinical data have proved the importance of CARD11 in DLBCL. Now there are few reports about the prognosis relationships among CARD11, NF-κB signaling transduction pathway and the immunophenotyping, the clinical features and biological characteristics of PGI-DLBCL.Objective:We detected the expression of CARD11, NF-κB subtypes p65, p50, CD10, Bcl-6, Mum-1, Bcl-2 and Ki-67 in primary gastrointestinal diffuse large B-cell lymphoma and associated with clinical and follow-up data. Our aim was to study and research systematically the relationships between CARD11 and NF-κB subtypes p65, p50 with the PGI-DLBCL immunophenotyping, the clinical features, biological behaviors of PGI-DLBCL. We also probed into screen the prognostic factors of PGI-DLBCL to find a more reliable molecular markers of prognosis. we can better provide a new theoretical basis for clinical application of targeted chemotherapy drugs of DLBCL.Methods:A total of 54 cases of PGI-DLBCL underwent operation in Shanghai Changzheng Hospital from May 1998 to June 2010 with complete clinical and follow up data involved in this study. The expression of CD10, Bcl-6, Mum-1 to immunophenotype and CARD11, NF-κB subtypes p65, p50 were detected on the formalin-fixed paraffin-imbedding tissues by immunohistochemistry. The SPSS statistical software were used in analyses on relationship between each factor expression and biological behavior, the apoptotic index Bcl-2 and the proliferation index Ki-67 of PGI-DLBCL. The significance of gene expression and survival period was determined by Kaplan Meier survival analyses and Log-Rank test, meanwhile the prognostic factors should be screened. Cox model were estabished according to multivariate analyses of survival data.Results: The positive expression rate of were detected as 28% in CD10, 69% in Bcl-6, 81% in Mum-1, 70% in CARD11, 72% in NF-κB/p65, 65% in NF-κB/p50 respectively. Of 54 primary gastrointestinal diffuse large B-cell lymphoma, 19 cases were classified as germinal center B-cell-like group(GCB), and 35cases asctivated B-cell like group(ABC). The expression rates of CARD11, NF-κB/p65 and NF-κB/p50 were 26.3%, 25.6% and 25.7% in GCB DLBCL, but were 73.7%, 74.4%and 74.3% in ABC DLBCL. The expressions rates of CARD11, NF-κB/p65 and NF-κB/p50 were higher in ABC-DLBCL than in GCB-DLBCL(P<0.05). Significant correlation was found between low positive expression and high positive expression of NF-κB/p65 and NF-κB/p50, with regard to CARD11(r=0.314, r=0.322, P<0.05). CARD11 expressed higher in the group of high-positive expression of NF-κB/p65 and NF-κB/p50. No significance correlation were found between the positive expression of CARD11 and gender, age, onset sites, clinical stage, depth of invasion, lymph node involvement, extranodal involvement sites, tumor size, LDH, ECOG, IPI, B symptoms of patients(P>0.05).The expression of NF-κB/p65 in PGI-DLBCL tissues was correlated with of extranodal involvement sites(group“≥2”> group“0~1”) (P<0.05), but was not correlated with gender, age, onset sites, clinical stage, depth of invasion, lymph node involvement, tumor size, LDH, ECOG, IPI, B symptoms of patients(P>0.05). The expression of NF-κB/p50 in PGI-DLBCL tissues was correlated with of clinical stage(group“Ⅲ/Ⅳ”> group“Ⅰ/Ⅱ”) (P<0.05), extranodal involvement sites(group“≥2”> group“0~1”) (P<0.05), LDH(group“higher”> group“normal”) (P<0.05), IPI(group“low risk”< group“low/intermediate”= group“high/intermediate”< group“high risk”) (P<0.05), but was not correlated with gender, age, onset sites, depth of invasion, lymph node involvement, tumor size, ECOG, B symptoms of patients(P>0.05). No significant correlation were found in Bcl-2 and Ki-67 of CARD11. Significant correlation was found between low positive expression and high positive expression of NF-κB/p65 and NF-κB/p50, with regard to Bcl-2(r=0.350, r=0.328, P<0.05). Significant correlation was found between low positive expression and high positive expression of NF-κB/p65 and Ki-67(r=0.271, P<0.05). No significance correlation were found in NF-κB/p50 and Ki-67(P>0.05). The gender, extranodal involvement sites, LDH, IPI, the immunophenotyping, expression of NF-κB/p65 were all correlated with survival time. The male group lived shorter than the female group; With the extranodal involvement sites, the“≥2”group lived shorter than the“0~1”group; the serum level of LDH at diagnosis had significantly lower survival rate; With the risk factors increasing, survival time of the lower levels(“low risk”and“low/intermediate”)were longer than the higher level(“high risk”and“high/intermediate”). Mean survival time of ABC is lower than GCB, NF-κB/p65 positive expression was longer than its negative expression.The other clinical agents were not correlated with survival time in PGI-DLBCL(P>0.05). The extranodal involvement sites and expression of NF-κB/p65 entered the final Cox model for prognosis of PGI-DLBCL.Conclusion:The expressions rates of CARD11, NF-κB/p65 and NF-κB/p50 were higher in ABC-DLBCL than in GCB-DLBCL, and the expression of CARD11 was statistically related with NF-κB/p65 and NF-κB/p50, which means CARD11 was consistend with NF-κB in ABC-DLBCL, and CARD11 as the important signal protein in the signaling pathway of NF-κB played a certain role in development of PGI-DLBCL. It means that, in clinical therapies, CARD11 could be a new potential target for treating PGI-DLBCL. The expression of NF-κB/p65 in PGI-DLBCL tissues was correlated with of extranodal involvement sites and the expression of NF-κB/p50 in PGI-DLBCL tissues was correlated with of clinical stage, extranodal involvement sites, LDH and IPI. Significant positive correlation were found in Bcl-2 of NF-κB/p65 and NF-κB/p50, so NF-κB could inhibit cell apoptosis and have positive correlation with the development and invasivion of PGI-DLBCL. Combining with the immunophenotyping, NF-κB could predict the development of PGI-DLBCL. The extranodal involvement sites, LDH, IPI, the immunophenotyping, expression of NF-κB/p65 could be the prognostic agents of PGI-DLBCL. The extranodal involvement sites and expression of NF-κB/p65 are major factors on prognosis of patients with PGI-DLBCL, and a high PI correlates with high risk and short survival time.更多还原