【作者】 赵菁；

【导师】 周韧；

【作者基本信息】 浙江大学 ， 病理学与病理生理学， 2010， 硕士

【摘要】 弥漫性大B细胞淋巴瘤(diffuse large B cell lymphoma,DLBCL)是目前最常见的成人非霍奇金淋巴瘤(non-Hodgkin’s Lymphoma,NHL)之一。长期以来,DLBCL作为NHL亚型却存在着明显的异质性,临床上表现为治疗反应的异质性、预后的异质性。DLBCL的异质性成为这一领域的研究热点。其发病机制中的高危因素包括免疫球蛋白基因重排以及体细胞超突变(SHM)。后者一旦发生异常,即可导致包括染色体易位、获得或者缺失等功能性突变的分子事件,扰乱B细胞稳态,可能导致B细胞增生,或凋亡受阻。体细胞超突变发生在B细胞分化的生发中心反应过程中,在此期间,Ig基因的V段产生高频点突变,致使B细胞产生高亲合力的IG分子。这个过程易受内、外因素(如个体遗传差异、病毒感染等)干扰而出现差异,构成了淋巴瘤发生的重要高危因素。值得注意的是,这种差异不仅仅发生在Ig基因中,也出现在其它的非Ig基因中,并诱导出变异。Pasqualucci等研究发现有45%的DLBCL病人在BCL-6、PIM1、c-MYC、PAX5、RhoH/TTF等基因存在异常超突变。同时,发现正常生发中心细胞(GCB)也发生BCL-6突变。这表明BCL-6可能是B-NHL中唯一的非Ig基因而具有与Ig基因相同超突变机制的基因。但是,目前BCL-6SHM的发生机制及对于DLBCL病人预后的意义尚不清楚。BCL-6 SHM是否与DLBCL某一亚型的发病相关?是否是肿瘤形成过程中“多步打击”中的一步?为了深入研究BCL-6 SHM与DLBCL分子亚型的相关性,我们对DLBCL病例进行了分子分型。WHO分类(2008版)中指出,在目前尚不能普遍开展基因表达模式分析(Gene expression profiling,GEP)的情况下,可用免疫组化检测CD10、BCL-6和MUM-1来判断是GCB型DLBCL(CD10+、BCL-6+/-、MUM-1-)或ABC型DLBCL(CD10-、BCL-6-/+、MUM-1+)。这种分类方式具有临床意义,患有GCB型DLBCL的患者往往预后好于患有ABC型DLBCL的患者。但是,同时也存在着一个问题:免疫组化由于多种因素的限制,对生发中心型阳性的判断准确率有不同程度的误差。为了克服免疫组化方法上的不足,我们期望可以找到一个指标有助于提高分类的准确性。基因表达谱研究表明,t(14;18)转位普遍发生于生发中心型DLBCL,而极少发生于非生发中心型DLBCL。另外,Huang等报道t(14;18)可确定具有GCB基因表达的DLBCL一个独特亚型。为此,本文利用免疫套餐标记,同时尝试结合t(14;18)易位检测对DLBCL进行联合分型,在此基础上进一步分析BCL-6非编码区突变与GCB亚型的相关性。t(14;18)易位通常发生在FL中,但是20%-30%的DLBCL也有发生。其实质是18号染色体上BCL-2基因与位于14号染色体上的免疫球蛋白重链发生转位。传统的细胞遗传学核型分析、FISH,和PCR可以检测这些基因重排,巢式PCR由于其敏感性较高,方便快捷而适用于研究。在本研究中,我们经筛选确定60例DLBCL为研究对象,进行两部分研究,在第一部分研究中,我们优化了DLBCL的分子分型。对60例DLBCL免疫组化分析,进行GCB和non-GCB分子分型。同时行巢式PCR检测t(14;18)易位。免疫组化结果显示,60例弥漫大B淋巴瘤中CD10的阳性率为21.7%(12/60),BCL-6的阳性率66.7%(40/60),MUM1的阳性率为71.7%(43/60),初步判断16例(26.7%)来源于GC,44例(73.3%)来源于non-GC。t(14;18)易位检测结果显示60例DLBCL中,共检出t(14;18)易位20例(33.3%),其中7例为主要断裂点MBR发生转位,13例为次要断裂点MCR发生转位。根据GCB型确定标准,t(14;18)+的DLBCL也归类为GCB型,结合免疫组化和转位检测结果,最终确定共26例均为GCB来源。第二部分内容是对BCL-6点突变进行分子生物学分析:PCR扩增BCL-6主要突变区,测序检测突变位点,并进行统计学分析。60例DLBCL标本中有12例发现有BCL-6基因碱基改变(5例结内DLBCL,7例结外DLBCL),碱基总突变率为20%。其中结内突变率为31.3%(5/16),结外突变率为15.9%(7/44)。突变类型多为碱基替换,也有移码突变。12个病例中,+363、+469和+397位点发生频率较高。统计学研究表明,BCL-6点突变多发生于生发中心型DLBCL中,可能与GC-DLBCL的发生发展相关。综上所述,基于以前的研究基础,我们联合免疫组化和t(14;18)易位检测GC型DLBCL取得了较为理想的效果,研究结果显示结合t(14;18)的检测可以提高GCB亚型的检出率,对于病人预后的判断具有重要意义,在后续研究中将进一步随访加以证实。此外,对60例DLBCL进行BCL-6超突变的分析显示突变多发生于GC型DLBCL中,可能是多步骤发生过程中的常见的分子事件。对GC型DLBCL的发生和发展可能具有重要作用。这一系列研究方法的建立为今后大样本分析奠定基础。更多还原

【Abstract】 Diffuse large B-cell lymphoma(DLBCL) is one of the most common non-Hodgkin’s Lymphoma(NHL) subtypes according to recent WHO classification of hematopoietic and lymphoid tumors(2008).However,DLBCL is characterized clinically by its heterogeneity,including the variable response to therapy and the highly variable prognosis,so the heterogeneity of DLBCL has become a hot spot focused by reasearchers.Ig gene rearrangment and SHM are not only two biological steps of germinal center reaction,but also risk factors of lymphomagenesis,the latter could contribute to many genetic abnormalities,including structural alterations(eg,balanced and unbalanced translocations),numerical changes(eg,aneuploidy),and single DNA point mutations,which disrupt the stability of B cell,lead to proliferation of B Cell or apoptosis blocked.Somatic hypermutation is a critical mechanism for the generation of diversity in the antibody repertoire.This process takes place in antigen-experienced B cells within the germinal center(GC),presumably the so-called centroblasts,and specifically targets rearranged Ig genes.During the germinal center reaction,the IgV-associated somatic hypermutation contribute to the IG molecular with high affinity.This process is easy to be disrupt by internal and external factors.Despite its high specificity for rearranged Ig genes,the somatic hypermutation mechanism was also shown to target non-Ig gene,such as the BCL-6,PIM1,c-MYC,PAX5,RhoH/TTF,and it targets BCL-6 in the normal GCB cells as well.BCL-6 is very special as a non-Ig gene but displays features of the IgV-associated somatic hypermutation mechanism,although the precise mechanism of BCL-6 SHM and its significance are poorly understood.In order to investigate BCL-6 SHM and its significance of tumorigenesis,we study the correlation between the mutation of 5’ noncoding region of BCL-6 gene and GCB subtype in diffuse large B-cell lymphoma,and subtyping for the DLBCL cases.According to the WHO classification(2008),Gene expression profiling of diffuse large B-cell lymphoma(DLBCL) has revealed biologically and prognostically distinct subgroups:germinal center B-cell-like(GCB),activated B-cell-like(ABC).but in the condition of routine work,we can use immunohistochemical stain to divide 2 major subgroups of DLBCL.But one has to keep in mind that the results of the immunohistochemical approach are highly variable,for the precision of method is limitied by both internal and external factors.In order to overcome it,we need a marker to improve the classification.The GEP studies reveals that t(14;18) is seen most often in the germinal center subtype of DLBCL and is generally associated with a more favorable prognosis.Huang et al reported that the t(14;18) defines a unique subset of diffuse large B-cell lymphoma with a germinal center B-cell gene expression profile.Therefore,we examined our DLBCL patients with both t(14;18) detection and IHC to determine the subtype,and further to analyse the correlation between the mutation of 5’ noncoding region of BCL-6 gene and GCB subtype in diffuse large B-cell lymphoma.The t(14;18)(q32;q21) is believed to play a crucial role in the pathogenesis of follicular lymphoma because it deregulates the expression of the antiapoptotic gene BCL2 by bringing it into proximity of the immunoglobulin heavy chain gene enhancer. The t(14;18)(q32;q21),with its associated BCL2 gene rearrangement,has also been detected in up to one third of cases of primary diffuse large B-cell lymphoma(DLBCL). Conventional cytogenetic karyotyping,FISH,and polymerase chain reaction techniques can reveal this genetic rearrangement,and nest-PCR is highly sensitive,easy to perform,and thus well suited for routine analysis of t(14;18) translocation.Our study included samples from 60 patients with DLBCL for two parts of research.In the first part,we optimized the molecular typing of DLBCL,t(14;18) detection and immunohistochemical staining were performed on samples from 60 cases with DLBCL,then divided them into GCB and non-GCB subtypes.12 of 60 expressed CD10,40 of 60 expressed BCL-6,and 43 of 60 expressed MUM1,cases were considered to be GC derived according to criteria of Alizadeh et al,about 16 of 60 of our DLBCL cases could be divided as GCB preliminarily.20 of 60 cases occured t(14;18) translocation,7 in major breakpoint region,13 in minor cluster region.Using the minimally acceptable criteria,26 of 60 cases were probably germinal centre derived.In the second part,Polymerase chain reaction(PCR),single-strand conformation polymorphism(SSCP) and direct DNA sequencing were used to identify mutations in the 5’ noncoding region of the BCL-6 gene.BCL6 mutations were detected in 20%of DLBCL cases(12/60),with a significantly higher frequency in the GC subgroups (34.6%) than in the non-GC subgroups(8.8%).BCL6 mutations in +363 and +469 sites happened frequently.Clear correlation between BCL6 mutation and GC subgroup was observed.In conclusion,a combination of immunohistochemical staining and t(14;18) translocation detection improved the molecular typing of DLBCL,it is significantly important for the prognosis,and the result need to be comfirmed by further studies.Our study also suggested that BCL6 mutations were detected in 20%of DLBCL cases,with a significantly higher frequency in the GCB subgroups than in the ABC subgroup.It is possible that BCL6 is important in the initial phase of transformation of DLBCL.BCL6 mutations may exert its oncogenic effects in the occurrence and development of DLBCL,The estabilishment of methods provide basis for further investigation.更多还原