【作者】 于宝华；

【导师】 施达仁； 周晓燕； 陆洪芬； 李小秋；

【作者基本信息】 复旦大学 ， 肿瘤学， 2009， 博士

【摘要】 目的:观察弥漫性大B细胞淋巴瘤(diffuse large B cell lymphoma,DLBCL)中PI3K/AKT/mTOR的活化状态及其临床病理相关性,从信号传导调控的角度阐述DLBCL的发病机理,以期为DLBCL的诊断以及预后评估提供新的分子指标;从PI3K催化亚单位的层次初步探讨DLBCL中PI3K/AKT/mTOR通路活化的机制;另外建立DLBCL裸鼠移植瘤模型,通过体内实验探讨AKT信号传导通路对DLBCL细胞生长及其生物学效应的影响,以期为DLBCL的治疗提供新的思路。方法:第一部分:收集复旦大学附属肿瘤医院病理科2002-2007年间新鲜DLBCL组织标本及相应石蜡组织75例,其中54例收集到随访资料;同时收集淋巴结反应性增生(reactive hyperplasia,RH)10例做对照。用免疫组织化学(immunohistochemistry,IHC)方法检测上述新鲜组织中pAKT、pmTOR的表达;同时用IHC法观察石蜡组织中Bcl6、CD10和MUM1的表达并据此将DLBCL分为生发中心B细胞样型(germinal center-B cell like,GCB)和non-GCB两型,运用TaqMan实时荧光定量逆转录聚合酶链反应(Real-time quantitativereverse transcription polymerase chain reaction,Real-time RT-PCR)技术检测Bcl6mRNA含量。分析pAKT、pmTOR在DLBCL及RH中表达的差异及其在DLBCL中的表达与分子分型、Bcl6表达及部分临床指标和预后的相关性。第二部分:选取第一部分中54例新鲜组织较多者,实时定量RT-PCR方法检测PI3K4个催化亚单位的mRNA表达;另外收集RH新鲜标本9例同时进行检测。分析4个亚单位相对表达水平的高低,及在DLBCL和RH中表达水平的差异,以及与pAKT表达的相关性。选择76例新鲜DLBCL组织标本,酚-氯仿法提取基因组DNA后针对PIK3CA基因突变热点部位所在片段(外显子9和外显子20)进行PCR扩增及基因突变的检测分析。第三部分:(1)无菌套管针抽吸法建立DLBCL裸鼠皮下移植模型,观察成瘤率和组织形态特点;用IHC法观察其免疫表型;用PCR扩增检测移植瘤组织和LY8细胞株的IgH克隆性重排和基因组DNA微卫星序列。(2)将荷瘤鼠(肿瘤大小均约200-400mm~3)随机分为3组,即对照组、低剂量LY294002组(0.1mg/kg)及高剂量LY294002组(0.5mg/kg),每组含10只裸鼠。每天给药连续10天。每周3次测量各移植瘤的体积(体积=1/2ab~2,其中a,b分别为移植瘤的长短径)。至第14天时牺牲裸鼠,HE染色观察移植瘤组织形态,IHC法检测Ki67表达并测定肿瘤细胞增殖活性,同时行Western blot方法检测AKT活化情况。并计算实体肿瘤质量抑瘤率和体积抑瘤率。(3)将荷瘤鼠(肿瘤大小均约600-800mm~3)随机分为4组,即6h处理组、18h处理组、24h处理组及48h处理组,每组4只裸鼠分别腹腔内注射对照(20%DMSO)、0.02mgLY294002、0.1mgLY294002或1.0mgLY294002。分别于给药一次后6h、18h、24h和48h牺牲裸鼠,采用流式细胞技术(flow cytometry,FCM)检测肿瘤细胞的细胞周期和细胞凋亡。(4)将荷瘤鼠(肿瘤大小均约200-400mm~3)随机分为4组,即单用LY294002(0.5mg/kg)组、单用阿霉素(doxorubicin,DOX)(5.0 mg/kg)组、LY294002(0.5mg/kg)与DOX(5.0 mg/kg)联合用药组以及对照组,每组含10只裸鼠。LY294002为每日给药连续10天,DOX为处理起始第1天和第3天给药。肿瘤体积观测、体积抑瘤率和质量抑瘤率计算、形态学观察及和细胞增殖活性检测大致同(2)。结果:第一部分:(1)pAKT和pmTOR在DLBCL中的阳性率分别为73.3%(55/75)和74.7%(56/75),二者的表达强度、阳性细胞分布基本一致。(2)non-GCB型DLBCL中pAKT和pmTOR的阳性率分别为82.5%(47/57)和84.2%(48/57),显著高于GCB型中的阳性率44.4%(8/18)和44.4%(8/18)(均p＜0.01)。(3)pAKT和pmTOR高表达组的Bcl6蛋白阳性率与mRNA水平均显著低于pAKT和pmTOR低表达或不表达者(均p＜0.01)。(4)pAKT和pmTOR阳性患者中乳酸脱氢酶(LDH)异常者高于阴性患者中的比例,差异达临界统计学意义(p=0.055)。pAKT和pmTOR的表达与性别、年龄、临床分期、KPS评分、B症状之间没有相关性(p＞0.05)。pAKT和pmTOR阳性患者的预后较阴性患者差,但差异没有显著性(p＞0.05)。第二部分:(1)4个催化亚单位在DLBCL中按照mRNA表达水平从高到低依次是PIK3CD,-B,-A和-G。其中PIK3CD表达水平略高于PIK3CB(p＞0.05),显著高于PIK3CA和PIK3CG(p＜0.05)。(2)PIK3CB的表达也显著高于PIK3CA和PIK3CG(p＜0.05),并且PIK3CB在DLBCL中的表达显著高于RH中(p＜0.05)。(3)pAKT阳性组中PIK3CB和PIK3CD的表达水平均显著高于pAKT阴性组(p＜0.05)。(4)76例DLBCL中仅有1例检出了PIK3CA基因的突变,突变率为1.32%。(5)该病例共有3个突变位点,其一为c.1634A＞C,发生在曾报道的热点部位之一;另两个突变位点是c.1658G＞C的颠换和其后单个碱基(T)的缺失(c.1659delT),导致移码突变,该两个突变均未见文献报道,为新发现的突变类型。第三部分:(1)运用LY8细胞株在裸鼠成功进行了人DLBCL细胞的种植,截至本次实验观察时已传至第9代,共移植裸鼠124只,其中114只成瘤,成瘤率达91.1%。每代移植瘤的生长特点均相似:于移植后约2周长出,随后即进入快速生长,约在3周左右长至1000mm~3,4周左右达3000mm~3大小。经病理形态学分析、IHC检测、基因重排检测及基因组DNA微卫星序列检测,表明与人DLBCL细胞相似,证实了移植瘤的稳定性和可重复性。(2)与对照组相比高剂量组LY294002显示出明显的抗肿瘤效应,且大致呈时间依赖性。高剂量LY294002组的肿瘤体积和质量与对照组相比均显著降低(p＜0.01);低剂量组降低不显著(p＞0.05)。与对照组相比低剂量组与高剂量组pAKT表达均显著降低,高剂量组尤其显著。(3)低剂量组和高剂量组LY294002肿瘤细胞增殖活性(Ki67阳性率)显著低于对照组,以高剂量组尤其显著(均p＜0.05)。(4)FCM检测发现6h和18h不同剂量组、24h低、中剂量组及48h中剂量组均显示肿瘤细胞的细胞周期S期细胞比例较对照略有降低而G1期细胞增加,提示出现G1期阻滞,但效果均不显著(p＞0.05)。24h时高剂量组和48h时低剂量组及高剂量组均显示出S期细胞比例明显降低(p＜0.05),提示出现显著的G1期阻滞。其中对细胞周期阻滞效果最显著的是48h时高剂量处理组。另外本试验也显示,LY294002促细胞凋亡的效果不明显(p＞0.05)。(5)与LY294002或DOX单用组及对照组相比,LY294002与DOX联合组荷瘤鼠较早地出现了对肿瘤生长的明显抑制。联合组的肿瘤体积抑瘤率显著优于任何单用组(p＜0.05);而质量抑瘤率显著优于LY294002单用组(p＜0.05),也优于DOX单用组,其差异达临界统计学意义(p=0.057)。通过检测Ki67的表达分析了LY294002对DLBCL肿瘤细胞增殖活性的影响,结果发现LY294002与DOX联用组的肿瘤细胞增殖活性显著低于对照组和任一药物单用组(均p＜0.01)。结论:1.AKT/mTOR信号传导通路活化在DLBCL发生中起重要作用,特别与Bcl6低表达或不表达以及non-GCB亚型密切相关,该信号通路有望成为部分DLBCL治疗的新靶点。2.在DLBCL中PIK3CD和PIK3CB均高表达并且与pAKT的表达显著相关,提示PIK3CD和PIK3CB可能在DLBCL中PI3K/AKT通路的活化中发挥重要作用。PIK3CB在DLBCL中的表达水平显著高于RH,提示其可能参与了DLBCL的发病机制。PIK3CA基因突变在DLBCL中发生率极低,提示其可能不是DLBCL中PI3K/AKT通路活化的主要机制。3.本研究成功建立了人DLBCL裸鼠移植瘤模型,为进一步体内研究提供了较理想的动物模型。利用该模型本研究表明LY294002在体内可以抑制AKT活化从而抑制DLBCL肿瘤的生长,并且主要是通过调节细胞周期而非细胞凋亡,同时可以抑制肿瘤细胞增殖活性。另外LY294002在体内也可以增加化疗药物DOX的肿瘤抑制作用。上述结果与本课题组前期体外实验结果相符,为DLBCL的治疗提供了新的理论依据和思路。本课题的创新性在于:1.从AKT/mTOR信号传导通路的角度探讨DLBCL的发病机制,目前国内尚无相关研究报道,国外仅有一篇相关报道,且未涉及该通路与分子分型及Bcl6表达等相关性的相关研究。本研究探讨了该通路的两个重要分子AKT和mTOR在DLBCL中的活化及其与DLBCL分型以及DLBCL中重要分子Bcl6表达的相关性,并且初步分析了该通路活化与临床指标以及预后的关系。2.关于DLBCL中PI3K/AKT通路的活化机制及DLBCL中PI3K 4个催化亚单位的表达水平目前国内外尚未见相关文献报道。本研究采用实时定量RT-PCR的方法从mRNA水平检测了4个催化亚单位的表达,并初步探讨各个亚单位表达与PI3K/AKT通路活化的关系。3.本研究成功建立了人DLBCL裸鼠移植瘤模型,为进一步体内研究提供了较理想的动物模型;并利用该模型对DLBCL中PI3K/AKT通路的生物学效应进行了研究。目前国内外尚未见相关研究报道。更多还原

【Abstract】 Objective:The aims of this study are to investigate the activation of PI3K/AKT/mTOR signal pathway in diffuse large B cell lymphoma（DLBCL） and its association with clinicopathologic characteristics,to elucidate the pathogenesis and development of DLBCL from the viewpoint of signal transduction,with the purpose of providing new markers for the diagnosis and prognosis of DLBCL;and further investigate the mechanisms of this pathway’s activation from the level of subunits of PI3K.In addition,we developed a mice model of DLBCL,to investigate the involvement of AKT pathway and its effects in DLBCL in vivo,which not only provided an animal model for the in vivo study of DLBCL,but also provided new therapeutic targets for the treatment of DLBCL.Methods:Part 1:（1） The frozen tissue samples and corresponding formalin-fixed paraffin-embedded samples of 75 DLBCL cases and 10 cases of reactive hyperplasia （RH） acquired from 2002-2007 were obtained from the Tumor Tissue Bank in the Department of Pathology,Cancer Hospital of Fudan University.Among the 75 DLBCL cases,follow-up data were available for 54 cases.（2） Immunohistochemistry（IHC） staining on frozen section was used to detect the expression of pAKT and pmTOR in the 75 DLBCL and 10 RH cases.IHC was performed on formalin-fixed paraffin-embedded sections to investigate the expression of Bcl6,CD10 and MUM1,according to which the DLBCL cases were divided into two subtypes,germinal center-B cell like（GCB） and non-GCB. Real-time quantitative reverse transcription polymerase chain reaction（Real-time RT-PCR） was performed to detect the mRNA expression of Bcl6 in the above cases. （3） The expression of pAKT and pmTOR in DLBCL and RH was compared and the association of these two molecules expression with Bcl6 expression,DLBCL subclassification,clinical biomarkers and the overall survival were analyzed.Part 2:（1） Real-time PCR was performed to investigate the mRNA expression of the 4 catalytic subunits of PI3K on 54 DLBCL cases from part 1 and 9 RH cases. Then the expression level of the 4 subunits was compared.The difference of their expression in DLBCL and RH and the association with pAKT expression were analyzed.（2） Genomic DNA of 76 DLBCL cases was extracted using phenol-chloroform and ethanol precipitation.Polymerase chain reaction（PCR） was performed to amplify the fragment with reported hotspots of mutation,and amplification products were then purified and directly sequenced.Part 3:（1） DLBCL-bearing mice model was first developed using human DLBCL cell line LY8.The hlstologic characteristics and the immunophenotype were examined.PCR amplification was performed to investigate the IgH cloned rearrangement and three microsatellite loci of xenografted tumor samples and LY8 cell line.（2） Tumor-beating mice were divided randomly into 3 groups when the tumor mass reached 200-400mm³:（ⅰ） LY294002（0.1mg/kg）,（ⅱ） LY294002 （0.5mg/kg）,（ⅲ） vehicle alone（control）.Each group consisted of 10 mice and treated each day continually for 10 days.Tumor size was measured 3 times per week.At the end of the experiments,the mice were humanly sacrificed and the tumors were excised and bisected.Western blot was used to detect the activation of AKT,and histological appearance was examined using hematoxylin-eosin（HE） staining and Ki67 staining was detected using IHC test and then proliferation index was analyzed accordingly.The weight inhibitory rate and the volume inhibitory rate were calculated.（3） DLBCL-bearing mice were divided randomly into 4 groups when the tumor mass reached 600-800mm³.Each group included 4 mice,which were administered intraperitoneally 0.02mg,0.1mg,and 1.0mg LY294002 and 20% DMSO,respectively.The 4 groups were sacrificed after treated for 6h,18h,24h,and 48h,respectively.Tumor masses were excised and cell cycle and apoptosis were detected using flow cytometry（FCM）.（4） DLBCL-bearing mice were divided randomly into the following 4 groups when the tumor mass reached 200-400mm³:（ⅰ） LY294002（0.5mg/kg,each day for 10 days）,（ⅱ） DOX(5mg/kg,the 1^st day and the 3^rd day),（ⅲ） DOX(5mg/kg,the 1^st day and the 3^rd day)+LY294002（0.5mg/kg,each day for 10 days）（ⅳ） vehicle alone（20%DMSO）.Each group consisted of 10 mice. LY294002 and vehicle were administered intraperitoneally and DOX was given intravenously.Tumor size measurement,histological appearance examination, proliferation index analysis,and weight inhibitory rate and the volume inhibitory rate calculation were performed according to（2）.Results:Part 1:（1） The expression of pAKT and pmTOR was 73.3%（55/75） and 74.7% （56/75）,respectively,and the expression of the two proteins was in consistent with each other.（2） The expression of pAKT and pmTOR in non-GCB group was 82.5% （47/57） and 84.2%（48/57）,respectively,which were significantly higher than that in GCB group,44.4%（8/18） and 44.4%（8/18）,respectively（both p＜0.01）.（3） The expression of Bcl6 protein and mRNA in pAKT and pmTOR high-expression group was significantly lower than that in low-expression group（both p＜0.01）.（4） The percentage of patients with abnormal LDH in pAKT and pmTOR positive groups was higher than that in negative groups,although it only reached borderline significance（both 10=0.055）.There was no relationship between the expression of pAKT and pmTOR and age,sex,stage,KPS and B symptoms（p＞0.05）.The overall survival of patients with negative pAKT was better than that with positive pAKT, although it did not reach statistical significance（p＞0.05）.Part 2:（1） Quantitative analysis using real-time PCR showed that PIK3CD expression was the most predominant among the 4 isoforms,with PIK3CB closely following it,while PIK3CG expression was the lowest.PIK3CD expression was significantly higher than PIK3CA and PIK3CG（p＜0.05）.（2） The PIK3CB expression was also significantly higher than PIK3CA and PIK3CG（p＜0.05） and its expression in DLBCL was significantly higher than that in RH（p＜0.05）.（3） Both PIK3CB and PIK3CD expression level were significantly associated with the pAKT expression,with high mRNA level of PIK3CB and PIK3CD in cases with positive pAKT expression（both p＜0.05）.（4） Only 1 out of 76 primary DLBCLs（1.32%） displayed mutations.（5） Three mutation points were found.One was a missense mutation（c.1634A＞C） which occurred in one of the reported hotspots.The other mutations,c.1658G＞C and c.1659delT,had not been hitherto reported in hematopoietic or solid tumors.Part 3:（1） We successfully developed DLBCL-bearing mice using human DLBCL cell line LY8,and it grew with high stability.By the time we analyzed,the mice had been steadily transferred to the 9^th generation;the total number of xenografted mice was 124,among which 114 mice were successfully transplanted with DLBCL,with the tumor formation rate remaining 91.1%.The grow characteristics of each generation were similar.Generally,the tumor mass appeared in about 2 weeks after inoculation,and then grew fast,and the tumor volume reached 1000mm³ in diameter in about 3 weeks and 3000mm³ in about 4 weeks.The histologic appearance,IHC detection,gene rearrangement detection,and Microsatellite DNA amplification of the xenotransplanted tumors and LY8 cell line showed that the histologic and genetic features,and Microsatellite DNA of xenotransplanted tumors were identical with that of the DLBCL cell line LY8.（2） Compared with the control group,high dose LY294002 group showed significant anti-tumor effect in a time-depended manner, and both the tumor volume and tumor weight in high dose group were dramatically reduced（p＜0.01）.The low dose group did not show noticeable anti-tumor effect （p＞0.05）.pAKT expression in low and high dose groups were significantly reduced compared with the control,with the high dose group being more dramatic.（3） Both the proliferation activity of low dose group and high dose group were dramatically lower than that of control group（both p＜0.05）.（4） The ratio of cells in S phase in each dose of 6h and 18h group was slightly reduced compared with the control,but the effect was not significant（p＞0.05）.The ratio of cells in S phase was significantly reduced in high dose of 24h group and the low and high dose of 48h group（p＜0.05）. In our study,48h is the best time period for LY294002 in cell cycle arrest.In addition,we showed that LY294002 did not induce cell apoptosis in this in vivo study（p＞0.05）.（5） The mice of LY294002 combined with DOX group showed significant inhibition of tumor growth much earlier than that of the LY294002 alone, DOX alone and control group.In addition,we showed that the tumor cell proliferation activity of the combined group was dramatically lower than that of control and LY294002 alone and DOX alone group（p＜0.01）.Conclusions:Part 1:PI3K/AKT pathway might play an important role in the development of DLBCL,and it was closely related to the low-or non-expression of Bcl6 and non-GCB subgroup.This pathway might serve as new target in the treatment of certain group of DLBCL.We have not found any related reports in China and only one abroad,which did not investigate the correlation of this pathway with DLBCL subtype and Bcl6 expression.Part 2:To the best of our knowledge,there was little is known about the mechanisms of PI3K/AKT pathway activation and the expression of the 4 PI3K catalytic isoforms in DLBCL.In the current research,we suggested that both PIK3CD and PIK3CB high expression might play important roles in the activation of PI3K/AKT pathway in DLBCL.PIK3CA mutations are infrequent in DLBCL, indicating that this mutation might not be a common route of frequent activation of the PI3K pathway in DLBCL.Part 3:We successfully developed a mice model of human DLBCL,providing a nice model for further investigation of DLBCL in vivo.We showed that LY294002 might inhibit the activation of AKT and the tumor growth of DLBCL-bearing mice, probably through the regulation of cell cycle instead of cell apoptosis.LY294002 also inhibited the proliferative activity of tumor cells.We also demonstrated that LY294002 might enhance the anti-tumor effect of DOX in vivo.更多还原