【作者】 刘杰；

【导师】 于金明；

【作者基本信息】 山东大学 ， 临床医学， 2013， 博士

【摘要】 研究目的：非霍奇金淋巴瘤(non-Hodgkin’s lymphoma, NHL)是一种发生于免疫系统的恶性肿瘤,也是目前世界上增长速度最快的恶性肿瘤,特别是近10年来增长速度显著增加。在美国每年大约66000例新增病例,18000例死亡病例；在中国,非霍奇金淋巴瘤发病率为3.5/10万,每年大约45000例新发病例,占恶性肿瘤发病第9位。流行病学研究表明,非霍奇金淋巴瘤具有家族聚集现象,近亲(尤其是兄弟姊妹或父母)中有血液/淋巴系统恶性疾病史者非霍奇金淋巴瘤发病风险可增加2-4倍,提示非霍奇金淋巴瘤的发病具有遗传易感性。同时研究也表明免疫功能失调是非霍奇金淋巴瘤的相关因素,提示免疫相关基因在非霍奇金淋巴瘤的发病中具有重要作用。因此,研究免疫相关基因的遗传变异背景对于阐明非霍奇金淋巴瘤的发病机制具有重要意义。本研究的主要内容包括以下三个部分：(1)对2002年1月至2010年12月山东省肿瘤医院收治的1248例非霍奇金淋巴瘤患者进行临床及病理特征分析；(2)研究免疫相关基因白细胞介素-10(Interleukin-10, IL-10)、肿瘤坏死因子-α (tumour necrosis factor-α, TNF-α)、淋巴毒素(lymphotoxin,ETA)以及细胞毒T淋巴细胞相关抗原-4(Cytotoxic T-lymphocyte antigen4, CTLA-4)的12个SNP位点与山东汉族人群非霍奇金淋巴瘤易感性的关系；(3)对与NK/T细胞淋巴瘤易感性密切相关的LTA+252A>G位点的SNP的功能进行初步研究。研究方法：(1)2002年1月至2010年12月在山东省肿瘤防治研究院收治的具有连续性诊断为非霍奇金淋巴瘤的患者收集包括性别、年龄、病理类型及免疫组化、临床分期、病变部位、诊断日期、家族史、个人史、治疗方式等临床资料进行计数或计量资料进行统计分析；(2)采用病例对照流行病学方法和候选基因法,对947例非霍奇金淋巴瘤患者和1044例相应健康对照抽取抗凝外周血,提取基因组DNA,采用聚合酶链反应-连接酶检测反应(polymerase chain reaction-ligation detection reaction, PCR-LDR)技术对IL-10((-3575T>A,-1082A>G,-819T>C以及-592A>C).TNF-α(-857C>T,-308G>A以及-238G>A)、LTA(+252A>G)和CTLA-4(-1661A>G,-318C>T,+49A>G以及CT60A>G)的12个SNP多态性位点进行基因分型检测,并采用logistic回归分析方法分析上述基因位点的多态性与非霍奇金淋巴瘤及其主要亚型(弥漫大B细胞淋巴瘤、滤泡性淋巴瘤、外周T细胞淋巴瘤和NK/T细胞淋巴瘤)易感性的关系；(3)采用酶联免疫吸附试验(ELISA)检测NK/T细胞淋巴瘤患者组和健康对照者组血浆LTA蛋白表达水平,并分析NK/T细胞淋巴瘤患者中携带不同LTA+252位点基因型与血浆LTA蛋白表达的关系,对LTA+252位点SNP的功能活性进行初步分析。结果：(1)1248例非霍奇金淋巴瘤患者临床及病理特征分析结果：①1248例非霍奇金淋巴瘤患者中,B细胞淋巴瘤807例(64.7%);T细胞淋巴瘤378例(30.3%),非霍奇金淋巴瘤未分类者63例(5.0%),比率最高的六种亚型分别为弥漫大B细胞淋巴瘤(40.9%)、NK/T细胞淋巴瘤(10.0%)、外周T细胞淋巴瘤(9.2%)、滤泡性淋巴瘤(6.4%)、粘膜相关结外边缘区B细胞淋巴瘤(5.4%)、前驱T淋巴母细胞淋巴瘤(4.5%)；②从性别比率来看,B细胞淋巴瘤患者男女比为1.4：1,T细胞淋巴瘤患者男女比为2.0：1；③非霍奇金淋巴瘤平均年龄为47.74±16.3(18～85岁),B细胞淋巴瘤平均年龄为50.7±15.2岁,T细胞淋巴瘤平均年龄为42.0±17.0岁；发病年龄高峰呈单峰,为50～59岁年龄段；④从病变部位来看,淋巴结内发病比例约为64%,结外为36%,结外非霍奇金淋巴瘤最常见部位依次为消化系统、鼻腔和韦氏环。(2)免疫相关基因IL-10、TNF-α、CTLA-4和LTA的12个SNP与非霍奇金淋巴瘤易感性关系研究结果：①对于总的非霍奇金淋巴瘤来说,未见上述基因多态性位点与总非霍奇金淋巴瘤的发病具有相关性。②将非霍奇金淋巴瘤分为B细胞性非霍奇金淋巴瘤和T细胞性非霍奇金淋巴瘤两大类时,TNF-α-857TT基因型与B细胞性非霍奇金淋巴瘤的发病有关；IL-10-1082AG基因型和G等位基因、IL-10-819CC基因型和C等位基因、LTA+252AA基因型与T细胞性非霍奇金淋巴瘤的发病有关(P<0.05)。③进一步对B细胞性和T细胞性非霍奇金淋巴瘤进一步亚型分析,IL-10-819CC基因型和C等位基因、IL-10-592CC基因型和C等位基因以及TNF-a-857TT基因型与滤泡性淋巴瘤发病有关(P<0.05)；IL-10-1082AG基因型和G等位基因、CTLA-4-1661AG基因型和G等位基因、CTLA-4-318CT基因型和T等位基因、CTLA-4+49G等位基因与外周T细胞淋巴瘤的发病有关(P<0.05); LTA+252AA基因型与NK/T细胞淋巴瘤的发病有关(P<0.05)。(3)与NK/T细胞淋巴瘤易感性密切相关的LTA+252位点SNP功能活性初步研究的结果显示,NK/T细胞淋巴瘤患者组血浆LTA水平明显高于健康对照组(P<0.05); NK/T细胞淋巴瘤组中,LTA+252AA基因型者血浆蛋白表达水平较之GG基因型患者蛋白表达明显增高,差异有统计学意义(391.2±85.6pg/ml vs.197.3±31.5pg/ml,P<0.05), LTA+252AG基因型者血浆蛋白表达亦有增高,差异具有临界性统计学意义(312.2±68.9pg/ml vs.197.3±31.5pg/ml, P=0.06)。结论：(1)1248例非霍奇金淋巴瘤患者临床及病理特征分析结论：本研究表明山东汉族人群NHL的临床及病理分布与亚洲人群基本相似,但与西方国家有显著性差异,具有NK/T淋巴瘤比例显著增高的特点。总之,B细胞性淋巴瘤在全世界范围内均具有较高的比例,而中国T细胞淋巴瘤的比例较西方国家显著增高。DLBCL在全世界范围内均具有最高的发病率,占全部NHL的25%-50%。FL和CLL/SLL在西方国家显著高于中国,而T细胞淋巴瘤(特别是PTL、NK/T和T-LBL亚型)的发病率在西方国家显著低于中国。因此我国汉族人群非霍奇金淋巴瘤不仅在发病率上与欧美国家有显著性差异,同时在疾病分类的分布上亦存在明显的不同。我国汉族人群非霍奇金淋巴瘤具有自己独特的临床及病理特征。该研究为山东地区NHL流行病学研究提供重要参考价值。(2)免疫相关基因IL-10、TNF-α、CTLA-4和LTA的12个SNP与非霍奇金淋巴瘤易感性关系研究结论：本研究提示存在于遗传背景上的免疫相关基因IL-10、TNF-α、LTA和CTLA-4的单核苷酸多态性可能与汉族人群非霍奇金淋巴瘤多种不同亚型的发病有关。其中,IL-10基因多态与滤泡性淋巴瘤的发病相关,IL-10和CTLA-4基因多态与外周T细胞淋巴瘤的发病相关,LTA基因多态与NK/T细胞淋巴瘤的发病具有一定相关。因此从免疫相关基因多态性与非霍奇金淋巴瘤易感性的关系研究为切入点,进行大样本病例-对照流行病学研究,极有可能发现非霍奇金淋巴瘤的致病关键基因并确定非霍奇金淋巴瘤的易感基因。(3)与NK/T细胞淋巴瘤易感性密切相关的LTA+252位点SNP功能活性初步研究的结论：位于LTA+252位点的遗传基因变异可能通过影响其相应的细胞因子LTA蛋白水平的表达从而影响NK/T细胞淋巴瘤的发生。意义：本研究首先对山东汉族1248例非霍奇金淋巴瘤患者的临床及病理特征进行分析,证实汉族人群非霍奇金淋巴瘤具有不同于西方的独特的临床及病理特征,同时在组建大规模的汉族人群非霍奇金淋巴瘤资源数据库基础上,采用PCR-LDR检测技术,对4个免疫相关基因的12个SNP位点进行分析,筛选并确定非霍奇金淋巴瘤及其主要亚型(弥漫大B细胞淋巴瘤、滤泡性淋巴瘤、外周T细胞淋巴瘤和NK/T细胞淋巴瘤)的免疫相关易感基因,对筛选出的易感基因进行初步的功能分析,本研究为进一步在全基因组范围内进行全基因组关联分析筛查非霍奇金淋巴瘤易感基因、明确非霍奇金淋巴瘤的发病机制以及揭示各种族间发病差异奠定良好基础。更多还原

【Abstract】 Objective:Non-Hodgkin’s lymphoma (NHL) is a cancer originated in lymphatic hematopoietic tissue. The incidence of NHL has increased during the last twenty years around the world, but etiological factors contributing to this phenomenon remain still largely unknown. There were66,120cases of newly diagnosed NHL in the United States, ranking as the sixth most common cancer. According to the published data from the2008Shanghai World Lymphoma Awareness Day, the incidence rate was3.5/100,000and the number of newly diagnosed cases was45,000every year, with more than20,000deaths, ranking from the10th to the9th in the malignant tumor incidence. Several large-scale studies have reported associations between family history of hematopoietic malignancy and lymphoma risk, it is likely that more common genetic variants may influence disease susceptibility. It also has been suggested that immune dysfunction may be the underlying basis of lymphomagenesis. Therefore, investigation of the inherited genetic variation background of immune-related genes is important for revealing and elucidating the pathogenesis of NHL. Based on the formation of large-scale Han people of NHL resources database, we detected12SNP loci of4immune-related genes using PCR-LDR technology, screened and identificated the immunity-related SNPs loci for NHL and its major subtypes (DLBCL, FL, PTL and NK/T lymphoma), we also made a functional analysis of the screened susceptibility SNP loci. Our study will lay a foundation for screening the NHL susceptibility genes in the genome-wide association studies and identify the NHL pathogenesis. The contents of this study includes three parts:(1)To investigate the clinical and pathological data of1248cases of NHL patients in Shandong Cancer Hospital from January2002to December2010.(2)To detect the association between four immune-related genes (Interleukin-10, tumour necrosis factor-a, lymphotoxin and cytotoxic T-lymphocyte antigen4) genetic variants and susceptibility to non-Hodgkin’s lymphoma in Shandong Chinese population.(3)To investigate the functional studies of LTA+252A>G SNP related to the susceptibility of NK/T cell lymphoma.Methods:(1)The clinical data of patients diagnosed with lymphoma in Shandong Cancer Hospital continuously from January2002to December2010were retrospectively analyzed. The clinical data of patients included gender, age, pathological type and immunohistochemistry, clinical stage, lesion location, date of diagnosis, family history, personal history. Significant differences between the proportions or the averages were analyzed using chi-square test or t test.(2) We genotyped12SNPs of the IL-10(-3575T>A,-1082A>G,-819T>C and-592A>C),TNF-α(-857C>T,-308G>A,-238G>A), LTA (+252A>G) and CTLA-4(-1661A>G,-318C>T,+49A>G, CT60A>G) using polymerase chain reaction-ligase detection reaction (PCR-LDR) analysis in947subjects with NHL and1044healthy controls. Odds ratio and95%confidence interval were determined by logistic regression for the associations between genotypes or haplotypes with the risk of NHL and subtypes (DLBCL, FL, PTL and NK/T)(3)The serum LTA protein expression level of patients with NK/T cell lymphoma and controls were detected using enzyme-linked immunosorbent assay (ELISA), the correlation between LTA+252polymorphism and plasma protein expression was analyzed.Results:(1)The pathological subtypes of the patients included in this study were classified according to the Revised European-American Lymphoma (REAL)/World Health Organization lymphoma classification scheme (2001).①There were807cases of B-cell lymphoma (64.7%);378cases of T-cell lymphoma (30.3%). Among them, the pathological types were mainly:diffuse large B cell lymphoma (DLBCL)(40.9%); extranodal NK/T-cell lymphoma, nasal type (NK/T)(10.0%); Peripheral T cell lymphoma, unspecified (PTL)(9.2%); follicular lymphoma (FL)(6.4%); extranodal marginal zone B cell lymphoma (MALT)(5.4%); precursor T lymphoblastic leukemia/lymphoma (T-LBL)(4.5%);②The male to female ratio of B-cell NHL patients (1.4:1) was significantly lower than that of T-cell NHL patients (2.0:1). ③The mean age of patients with B-cell NHL of50.7±15.2was significantly higher than that of the patients with T-cell NHL of42.0±17.1;④In this study,64.0%lesions were lymph nodes and36.0%were extranodal sites. The most common sites of Extranodal were digestive system, nasal cavity and Waldeyer’s ring.(2) No significant difference was seen between these12gene polymorphisms (SNPs) and total NHL. TNF-α-857TT genotype was associated with increased risk of B-NHL; IL-10-1082AG genotype and G allele, IL-10-819CC genotype and C allele, LTA+252AA genotype were associated with increased risk of T-NHL. When the main pathological subtypes of NHL was analyzed, IL-10-819CC genotype and C allele, IL-10-592CC genotype and C allele, TNF-α-857TT genotype was associated with FL; IL-10-1082AG genotype and G allele, CTLA-4-1661AG genotype and G allele, CTLA-4-318CT genotype and T allele, CTLA-4+49G allele was associated with PTL; the LTA+252GG genotype was associated with NK/T lymphoma.(3) The analysis of functional activity of LTA+252A>G SNP showed that the plasma LTA protein level was significantly elevated in NK/T cell lymphoma patients than in controls (P<0.05). In the NK/T cell lymphoma patients, the plasma LTA protein level of individuals with AA genotype was significantly higher than those with GG genotype(391.2±85.6pg/ml vs.197.3±31.5pg/ml, P<0.05); The LTA protein level of individuals with AG genotype was boardline higher than those with GG genotype (312.2±68.9pg/ml vs.197.3±31.5pg/ml, P=0.06)Conclusion:(1) This study suggests that the clinical and pathological distribution of NHL in Shandong province, China, is consistent with that for Asian populations, but significantly different from that in the Western countries, with higher proportion of NK/T lymphoma in this region. Overall, the B-cell lymphoma accounted for a high proportion worldwide, the T-cell lymphoma in China was however higher than that in European and American countries. DLBCL is the pathological subtype with the highest rate of incidence all around the world, accounting for25%-50%of NHL. The incidence proportions of FL and CLL/SLL in the United States, the Netherlands and Chinese-Americans were significantly higher than those in China. T-cell lymphoma (especially PTL, NK/T, and T-LBL) in European and American countries was significantly lower than that in China. Therefore NHL has its unique clinical and pathological features in Chinese Han population.(2)The inherited genetic variations in the immune-related genes IL-10, TNF-α, LTA and CTLA-4loci may influence risks of different subtypes of NHL in the Chinese population. The IL-10gene polymorphism was associated with FL, the IL-10and CTLA-4gene polymorphism was associated with PTL lymphoma and the LTA gene polymorphism was associated with NK/T cell lymphoma. Therefore, from the the breakthrough point that relationship between the immune related genes polymorphism and susceptibility of NHL, a large case-control study is very likely to discover the key genes of NHL pathogenesis and to determine the susceptibility genes of NHL.(3) The inherited LTA+252A>G site genetic polymorphisms is correlate with the LTA protein expression and may affect the NK/T cell lymphoma pathogenesis.Summary:Our results showed that genetic variation in the immune-related gene IL-10, TNF-α, LTA and CTLA-4has a certain correlation with the different risks of NHL subtypes.Significance:This study analyzed the clinical and pathological data of1248patients diagnosed with NHL in Shandong Cancer Hospital continuously from January2002to December2010, and proved that NHL has its unique clinical and pathological features in Chinese Han population. Then, based on the previous work, we genotyped12SNPs of the IL-10,TNF-α, LTA and CTLA-4genes using PCR-LDR method and analyzed the correlation between these genotypes and haplotypes and the risk of NHL and subtypes (DLBCL, FL, PTL and NK/T). Further, we made a functional analysis of LTA+252A>G SNP in NK/T cell lymphoma. Our study established a good foundation for the next step to research the genome-wide linkage studies screening susceptibility genes for NHL in Chinese population and to elaborate the pathogenesis of non-hodgkin’s lymphoma.更多还原