【作者】 李慧灵；

【导师】 赵彤；

【作者基本信息】 南方医科大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 研究背景与目的1985年Stein等首次描述间变性大细胞淋巴瘤(anaplastic large cell lymphoma,ALCL),并确认为一种新的淋巴瘤类型。ALCL是一种T细胞淋巴瘤,以CD30(肿瘤坏死因子受体超家族成员8,TNFRSF8)表达为特征,在成人非霍奇金淋巴瘤中比例小于5%,但在儿童中发病率较高,占儿童非霍奇金淋巴瘤的40%。原发系统型ALCL病理特征主要由淋巴样细胞组成,细胞较大,胞质丰富,胞核经常成马蹄样或胚胎样。原发系统型ALCL包括两个亚型:ALK(+)型和ALK(-)型。ALK(间变性淋巴瘤激酶,anaplastic lymphoma kinase)完整蛋白质分子是一个200kDa的受体酪氨酸激酶(receptor tyrosine kinase,RTK),由2p23染色体区段编码,拥有胞外区,穿膜区和胞内区。全长ALK与白细胞酪氨酸激酶高度同源,都属于胰岛素受体超家族。虽然ALK首先在造血系统肿瘤中被发现,但是ALK基因从未检测到在正常造血组织有表达。ALK在正常组织的功能迄今为止仍不清楚。在ALCL中出现的是由染色体易位t(2;5)(p23;q35)编码的NPM-ALK融合蛋白,2p23上的ALK基因易位至5q35的NPM(核磷酸蛋白)基因旁并被后者激活而高表达,ALK激酶呈激活状态可能是ALCL成瘤的关键机制,也是部分ALCL特征性的遗传学指标。融合蛋白长度80kDa,相应位置为全长ALK的胞内区部分,其N末端与NPM(phosphoprotein nucleophosmin)结合,NPM执行二聚体功能,结构性激活ALK的激酶活性和其致瘤性。NPM-ALK是一个“停靠蛋白质”,可以依次偶联有丝分裂、抗凋亡、粘附及DNA修复能力等多条信号传导途径。PL-C,P13K,STATs,和Src是NPM-ALK的重要下游靶标,对细胞有着促有丝分裂和抗凋亡的作用,是一个受到广泛认可的致瘤性酪氨酸激酶。ALK与ph染色体形成的致瘤性BCR/ABL酪氨酸激酶同源,现在针对ABL酪氨酸激酶的研究进展较快,其特异性抑制剂STI571已经在临床上成功治疗了BCR/ABL(+)的白血病患者。因此ALCL倍受关注,研究的焦点一部分集中于探讨该类染色体易位产生的酪氨酸激酶在肿瘤的发生发展中所起作用,但是ALK在原发系统型ALCL中,除了作为致瘤性酪氨酸激酶对ALCL的发生有重要的作用外,还在预后预测中有着比较特殊的地位,大部分的报道认为,ALK(+)是ALCL中最重要的预后指标,在美国、欧洲、日本的一系列研究中,ALK(+)与预后良好有关;因为ALK(+)ALCL对治疗的反应良好,接受经典CHOP化疗方案后完全缓解率高,少数的复发病人仍保持着对化疗敏感。尽管大多数研究者在临床研究中得到了ALK与ALCL良好预后具有正相关关系的结果,但ALCL中ALK对预后影响的机制目前并未得到良好诠释。因此本研究从ALCL病人临床病情、诊治过程、生存随访入手,首先验证ALK(+)ALCL和ALK(-)ALCL对治疗的不同反应性及ALK对预后产生的影响,然后从可能与ALK有关的机体免疫、肿瘤转移、侵袭、粘附、凋亡因子中寻找ALK对ALCL预后良好影响的可能机制与信号传导途径,并在细胞株中用不同方法抑制ALK的酪氨酸激酶活性表达后,实验验证这条信号传导途径对预后相关因子的影响,为ALCL预后判断及靶向治疗提供理论与实验依据。方法1 ALCL病人的病情与生存时间随访及ALK与预后的关系本部分对34例ALCL进行了病情和生存状况随访,其中4例病人失访。随访资料主要包含以下字段:一般信息(病案号、病理号、姓名、年龄、性别)和临床病情与生存状况信息(发病部位、是否有骨髓侵犯、起病时是否有B症状、治疗方案、治疗结果、肿瘤临床分期、IPI评分、生存时间、生存状态)。采用免疫组织化学染色和荧光原位杂交技术分别在蛋白水平和基因水平检测ALCL组织中的ALK蛋白和染色体2p23区段断裂。应用SPSS11.5统计分析软件进行统计分析,生存率采用寿命表法,单因素生存分析采用Kaplan-Meier法分析,Log-rank检验。各因子间相关分析采用卡方检验。2 ALCL临床病理组织内相关预后因子的筛选及与ALK相关预后信号传导途径推测采用免疫组织化学方法检测并筛选ALK在ALCL组织内影响的侵袭,粘附,凋亡相关因子,运用免疫荧光检测共定位的细胞内蛋白,应用PCR-SSCP分析P53的第5、6、7、8外显子突变情况,运用SPSS11.5统计分析软件进行统计分析,单因素生存分析采用Kaplan-Meier法分析,Log-rank检验,各因子间相关分析采用卡方检验,从中筛选与ALK和预后有关的因子,以期发现在ALCL组织内ALK对预后产生影响的信号传导途径。3 ALCL细胞株内抑制ALK酪氨酸激酶活性的研究及ALK相关预后信号传导途径验证在利用酪氨酸蛋白酶抑制剂除莠霉素A作用于ALCL细胞株karpas299以减弱ALK的酪氨酸激酶作用以及因AS干扰引起ALK表达降低这两种造成ALK表达减弱的情况下,检测组织中筛选出的信号传导途径分子。以台盼蓝拒染法和流式细胞检测仪检测药物作用前后ALK的酪氨酸激酶活性变化以及细胞凋亡和细胞周期阻滞情况;对作用前后相应信号传导途径蛋白进行免疫细胞化学染色分析(对半衰期较短的P53蛋白采取蛋白酶抑制剂处理延缓降解,并用PCR-SSCP分析从基因水平分析有无突变);对研究中关键性的ALK和cleaved caspase3蛋白进行Western Blotting半定量分析。结果1 ALCL病人ALK表达与临床病情、生存时间随访的关系ALK(+)ALCL病人预后较ALK(-)ALCL预后好。临床病情与生存分析发现与预后与ALK均相关临床因子有:年龄、化疗后完全缓解情况、临床肿瘤分期和IPI。2 ALK与侵袭粘附及凋亡通路因子关系及其可能影响预后的信号传导途径根据临床分析结果,选择有关因子在组织中进行了研究:CD44及其突变体、MMP2、TIMP1、nm23、CD56、survivin、Bax、bcl-2、P53、MDM2、HSP90α、细胞色素C和Cleaved Caspase3。经过对上述因子的初筛,与ALK有关的因子有:HSP90α与ALK在细胞内共表达,MDM2过表达、bax表达、细胞色素C表达、cleaved caspase3的表达与ALK表达成正相关,P53在ALK阳性的ALCL组织内未见突变,bcl-2与ALK表达负相关,且上述因子均与预后有关,根据这些因子中存在的上下游调节关系或相互影响的关系,可能组成一条ALK影响ALCL预后的信号传导途径。3 ALCL细胞株karpas299除莠霉素A抑制ALK酪氨酸激酶活性与预后相关因子变化除莠霉素A作用karpas299细胞后,karpas299细胞酪氨酸激酶活性下降,细胞凋亡率上升,细胞周期在G1期阻滞,并随药物浓度梯度和时间作用梯度的加大而升高;Hsp90α、MDM2表达减弱,野生型P53、bax、细胞色素C与cleaved caspase3表达增强。表达变化与对照组相比差别明显。4 karpas299细胞中ALK表达因AS干扰受抑制后对除莠霉素A的反应与预后相关因子变化对因AS干扰引起ALK表达降低的karpas299细胞研究发现,ALK受其他因素影响而表达降低后,对除莠霉素A的反应降低,除莠霉素A引起的凋亡较karpas299细胞明显减弱。同样的,MDM2,P53,bax,细胞色素C,cleaved caspase3等相关因子在药物作用后的表达变化较未做干扰的karpas299细胞程度减轻。表达变化与对照组相比差别明显。结论1 ALK(+)ALCL病人预后优于ALK(-)ALCL预后;2 ALCL临床预后相关因子主要有:发病年龄、完全缓解率、临床肿瘤分期和IPI评分。ALK(+)ALCL病人发病年龄轻,临床肿瘤分期较早和IPI评分较低,完全缓解率较高。3 ALCL临床预后相关因子对应组织因子表达为:ALK(+)ALCL:ALK、Hsp90α、MDM2过表达;Bax、细胞色素c、cleavedcaspase3有表达或强表达;bcl-2弱表达或无表达;无P53突变型蛋白表达。ALK(-)ALCL表达情况则与ALK(+)ALCL相反。4抑制ALK酪氨酸激酶活性后,ALK(+)ALCL细胞株中组织因子表达变化:ALK、Hsp90α、MDM2表达减弱;P53野生型蛋白出现表达;Bax、细胞色素c、cleaved caspase3表达增强。表达变化与对照组相比差别明显。根据临床病理与生存分析,组织学研究和细胞学研究综合分析表明,在ALK(+)ALCL中ALK通过这样一条可能信号传导途径促进肿瘤发生:染色体易位造成ALK的过度表达,依附Hsp90保证其蛋白活性,发挥其酪氨酸激酶活性激活ras途径,造成MDM2过表达,进而抑制了ALCL内野生型p53的表达,但是由于野生型p53的微量活性存在,其调节的下游分子仍然部分的发挥作用。因此通过抑制ALK进而抑制MDM2表达,可使野生型P53表达增强,其下游促凋亡分子Bax高表达,而抑制凋亡分子bcl-2表达减弱。Bax促进了细胞色素c从线粒体的释放,最终引起凋亡执行分子caspase3的激活,细胞趋向于发生凋亡。在这条途径中P53仅为MDM2抑制,未突变或仅为一条等位基因突变,野生型P53蛋白在被增强表达后起到促进细胞凋亡的作用,因此使ALK(+)ALCL对化疗有着良好的反应性,本研究认为这可能是其良好预后的一种机制。本研究创新之处将ALCL临床病情发展变化、生存分析、肿瘤组织学与细胞学结合探讨,对ALCL中ALK影响预后的可能机制进行了研究,为将来ALCL及其他涉及染色体易位产生致瘤性酪氨酸激酶的肿瘤更好的治疗提供理论依据。更多还原

【Abstract】 BACKGROUND & OBJECTIVEAnaplastic large cell lymphoma(ALCL) is a subtype of T-cell non-Hodgkin’s lymphoma characterized by the expression of CD30 antigen(also known as tumor necrosis factor).Primary systemic ALCL has a peak incidence in childhood, accounting for approximately 40%of NHL cases diagnosed in pediatric patients, whereas it accounts for＜5%of NHL in adults.The common type is composed of large,pleomorphic,atypical tumor cells with an abundant grey-blue cytoplasm.Their nuclei are large,horseshoe- or kidney-shaped,and sometimes have pseudonuclear inclusions(so-called doughnut cells).About half of these lymphomas have the t(2;5)(p23;q35) translocation and express a hybrid protein comprised of the anaplastic lymphoma kinase(ALK) and the nucleolar phosphoprotein nucleophosminutes (NPM).Additional translocations are rare in which ALK is fused to other partners. Half of ALCL do not express ALK or other recurrent translocations.Full-length ALK have been shown as novel,putative receptor tyrosine kinase (RTK) of the insulin receptor family,encoded by chromosome 2p23,and comprised of extracellular,transmembrane,and cytoplasmic domain.The most closely related kinase to ALK is leukocyte tyrosine kinase(LTK),which lacks a sequence corresponding to the amino terminal half of the ALK extracellular domain.The extracellular domain and protein kinase domain of LTK possess 50%and 78%amino acid identity,respectively,to the corresponding regions of ALK.These two kinases comprise a new subfamily of the insulin receptor family.Although ALK was originally identified in hematopoietic neoplasm anaplastic large cell lymphomas (ALCL),ALK expression was never detected in normal hematopoietic tissue. However ALK is expressed preferentially in neurons of the central and peripheral nervous systems at late embryonic stages.ALK function in normal tissue is still an enigma.In ALCL the cytoplasmic domain of ALK is fused to the amino terminal half of nucleophosmin(NPM) to generate a fusion gene product,p80NPM-ALK(Morris et al.,1994).NPM is a multifunctional protein that plays a role in protein shuttling between the cytoplasm and nucleus(Borer,at al.,1989) and in cell cycle-dependent entrosome duplication(Okuda et al.,2000).Previous studies have shown that the NPM portion of p80NPM-ALK mediates oligomerization.The t(2;5)(p23;q35) translocation creates the fusion gene NPM-ALK(p80) that encodes a product with tyrosine kinase activity believed to play an important role in development of anaplastic large cell lymphoma(ALCL).NPM-ALK acts as a docking protein for a signalosome which in turn couples the fusion protein to signalling pathways with mitogenic,antiapoptotic and possibly DNA repair capabilities.NPM-ALK induces oncogenic signals by autophosphorylation at multiple Tyr residues,followed by recruitment of a signalosome:an ensemble of proteins containing docking SH2 or PTB domains that couples the fusion protein to downstream signalling pathways. Investigations based largely on cell line data suggest that the NPM-ALK signalosome stimulates pathways regulating mitogenesis and survival,involving the phosphoinositide 3-kinase(PI 3-kinase) and Ras/MAP kinase pathways,and the STAT family of transcription factors. ALK can function as an oncogene via dysregulation of its tyrosine kinase regions in NPM-ALK(p80) much as bcr-abl tyrosine kinase.Tyrosine kinase inhibitors have been considered as antineoplastic agents.The recent studies demonstrated utilization of signal transduction inhibition as shown by STI-571,which selectively inhibits the activation of the bcr-abl tyrosine kinase for successful treatment of chronic myelogenous leukemia(CML).Therefore much attention was paid to ALK in ALCL although it is rare in human carcinoma.ALK in ALCL is known as an oncogenic protein.However,ALK protein expression is an independent predictor of survival and serves as a useful biologic marker of a specific disease entity within the spectrum of ALCL.Patients with ALK(+)ALCL are reported to have a better prognosis than patients with ALK(-)ALCL.Because the mechanisms for this survival difference are unknown and the prognostic factors that are useful for predicting survival remain unclear,we investigated the hypothesis that pathways of expansion,invasion,adhesion or apoptosis may be involved.We therefore collected the clinical features and survival time and attempted to identify the clinical and pathological factors of prognostic importance and then we assessed expression levels of the expansion,invasion, adhesion and anti-apoptotic proteins in ALCL tissue and identify them in Karpas 299 cell line using tyrosine kinase inhibitors to find a possible mechanism pathway of ALK effects to the prognosis in Anaplastic Large cell lymphoma.METHODS1 the clinical features and survival time follow-up in ALCL and the relationship between ALK and the prognosisThe study group included 34 ALCLs accessioned at Southern Medical University affiliated Nanfang Hospital,Fouzhou General Hospital and Shenzhen Nanshan Hospital between 1999 and 2006..The diagnosis of ALCL was based on criteria specified by the WHO classification.Cases of primary cutaneous ALCL were excluded.4 patients were totally lost contact information and lost to follow-up.FISH and immunocytochemistry was used to evaluate NPM-ALK in ALCL tissues on genome and protein levels,respectively.Survival analysis was based on life tables and Kaplan-Meier.Statistical comparison of ALK and other clinical parameters was based on the crosstabs analysis (Microsoft SPSS 11.5).2 screening possible pathway through which ALK may effect on prognosis in ALCLImmunocytochemistry was used to screening possible pathway through which ALK may effect on prognosis in ALCL.Immunofluorescence was used to analyze co-localization of ALK and Hsp90αproteins.PCR-SSCP analysis was used to detect P53 mutation of 5,6,7,8 exons.Survival analysis was based on life tables and Kaplan-Meier.Statistical comparison of ALK and other intracellular pathway parameters was based on the crosstabs analysis(Microsoft SPSS 11.5).3 identify the pathway found in the 2 chapter in Karpas 299 cell lineTyrosine kinase inhibitor Herbimycin A was used to inhibit the ALK tyrosine kinase activity in Karpas 299.Immunocytochemistry,trypan blue and Flow cytometry was used to detect the tyrosine kinase activity,apoptosis,and apoptosis-related factors.P53 protein was protected by protease inhibitor PMSF. PCR-SSCP analysis was used to detect P53 mutation of 5,6,7,8 exons in cells. Western Blotting electrophoresis was used to semi-quantitative analysis.RESULTS1 Relationship between ALK and clinical condition and survival time:Patients with ALK(+)ALCL had a better prognosis than patients with ALK(-)ALCL.Clinical parameters related both ALK and prognosis are:age;CR;clinical tumor stage;IPI.ALK(+)ALCL patients were younger than ALK(-)ALCL patients that means better basic condition presented in ALK(+)ALCL patients.ALK(+)ALCL patients had earlier clinical tumor stage and lower IPI than ALK(-)ALCL patients that means inferior invasion power presented in ALK(+)ALCL patients.ALK(+)ALCL patients had higher CR rate than ALK(-)ALCL patients that means better reactiveness to chemical therapy presented in ALK(+)ALCL patients.2 Pathway screening related to ALK and prognosis:After the screening of the possible pathway ALK effects on prognosis in ALCL tissue,CD4,CD5,CD44、MMP2、TIMP1、nm23、CD56、survivin、Bax、bcl-2、P53、MDM2、Hsp90α、cytochrome C and Cleaved Caspase3 were selected in our research.Hsp90αwas co-localized with ALK in ALK(+) ALCL tissue.MDM2 over expression and ALK expression did not have significant difference.P53 did not mutate in all ALK(+) ALCL tissues and mutated in 2 ALK(-) cases of ALCL.Bax expression and ALK expression did not have significant difference.Bcl-2 negative expression and ALK expression did not have significant difference.Cytochrome C and cleaved caspase3 expression and ALK expression did not have significant difference.3 Pathway reaction to Herbimycin A in Karpas 299Cell tyrosine kinase activity of ALK decreased and apoptosis increased after Herbimycin A action in Karpas 299 cells.Cell cycle was arrested in G1/S.ALK, Hsp90αand MDM2 expression decreased after Herbimycin A action.Wild type P53 protein,Bax,Cytochrome C and cleaved caspase3 expression all increased after Herbimycin A action.4 Pathway reaction to Herbimycin A in Karpas 299 with ALK downregulation by AS RNA interferencePathway reaction to Herbimycin A in Karpas 299 with ALK downregulation by AS RNA interference was weaker than that in Karpas 299.Cell tyrosine kinase activity of ALK decreased and apoptosis increased after Herbimycin A action but were in lower degree than that in Karpas 299.CONCLUSIONALK(+) ALCL has a better prognosis than ALK(-) ALCL.The clinical prognosis factors were age,;CR;clinical tumor stage;IPI.There may be such a pathway which induces the tumorigenesis of ALCL but probably be reason of the well prognosis of ALK(+) ALCL:Chromosome translocation t(2;5) lets hybrid protein NPM-ALK over expression.Hsp90 protects ALK and ALK can execute its receptor tyrosine kinase activity and stimulates Ras pathway.Ras pathway can enhances MDM2 expression and MDM2 can inhibit wild type P53 protein.P53 protein is inhibited by overexpression MDM2 but still has tiny effect on its downstream factors.When ALK tyrosine kinase activity was inhibited by Herbimycin A,Hsp90 loses its client and expresses weaker.ALK’s effect on ras pathway weakened and MDM2 expression is downregulated.Wild type P53 protein expresses after MDM2 expression is downregulated and effect of P53 on its downstream factors began manifest.Bax protein is upregulated by P53 and promotes Cytochrome C releasing from chondrosome.Caspase 3 is stimulated and apoptosis happens.更多还原