【作者】 王珂；

【导师】 刘艳艳；

【作者基本信息】 郑州大学 ， 内科学硕士（专业学位）， 2021， 硕士

【摘要】 背景和目的:弥漫大B细胞淋巴瘤(Diffuse large B-cell lymphoma,DLBCL)是来源于成熟B淋巴细胞的恶性肿瘤,是非霍奇金淋巴瘤(non-Hodgkin lymphoma,NHL)中最常见的类型,占成人NHL的30%～40%。DLBCL几乎可发生于人体任何部位,包括淋巴器官(韦氏环、脾脏、胸腺和淋巴结)和粘膜相关淋巴组织,常见的结外受累部位有头颈部的鼻腔、窦腔、软腭、涎腺和甲状腺,胸部的乳腺、肺和胸膜,腹部的胃、肠、肝脏、胰腺、肾和肾上腺,盆腔的子宫、卵巢和睾丸,中枢神经系统的脑、脑膜和脊髓,以及皮肤及软组织、骨骼和骨髓等。国际预后指数(International prognostic index,IPI)评分是评估DLBCL患者预后最常用的工具,包括年龄、分期、结外病变数目、乳酸脱氢酶(Lactic dehydrogenase,LDH)水平和美国东部肿瘤协作组(Eastern Cooperative Oncology Group,ECOG)行为状态,多个结外受累部位数(≥2个)被认为是不良预后指标之一。越来越多的研究表明,特定的受累部位也与预后相关,美国国立综合癌症网络(National Comprehensive Cancer Network,NCCN)IPI评分中,中枢神经系统、骨髓、肝脏、胃肠道、肺受累被认为是预后不利因素。我们的前期研究也发现累及韦氏环预后较好,累及中枢、骨髓、乳腺、肾上腺预后则较差。但是,多个结外受累数目和不同结外受累部位预后不良的分子机制和潜在的精准治疗方案尚不清楚。DLBCL的高度异质性导致现有的临床、形态学和免疫表型仍然不能满足临床精准诊断和治疗的需求,但随着高通量二代测序技术的发展,已有一些基因被发现在淋巴瘤中高频发生,提示这些突变的基因可能参与了淋巴瘤的发生发展,有希望被应用于淋巴瘤的诊断、分型、预测预后,甚至指导临床治疗决策。本研究通过检测和分析淋巴瘤相关驱动基因异常,初步探讨DLBCL患者中结外受累与淋巴瘤驱动基因异常的相关性,旨在揭示多个结外受累数目和不同结外受累部位预后不良的分子机制和潜在的精准治疗方案。资料和方法:收集2006年4月至2020年9月在我院收治的244例弥漫大B细胞淋巴瘤患者肿瘤组织标本,二代测序方法检测32个热点驱动基因突变。应用SPSS 22.0版本对数据进行统计分析,计数资料采用百分数(%)表示,组间分析选择双侧χ~2检验或Fisher精确概率法,Phi系数表示关联强度。多因素分析采用logistic回归法。生存分析采用Kaplan-Meier法,绘制生存曲线,并行Log-rank检验,P<0.05认为有统计学差异。结果1、244例DLBCL患者中发生结外部位受累163例,占总人数的66.8%(163/244)。其中无结外部位受累81例(81/244,33.2%),单个结外部位受累118例(118/244,48.4%),多个结外部位受累(≥2个)45例(45/244,18.4%)。中位随访时间20.4个月,中位无进展生存时间(progression-free survival,PFS)淋巴结单独受累患者27.0个月,单个结外部位受累患者53.6个月,多结外部位受累患者15.6个月(P=0.138);中位总生存时间(overall survival,OS)淋巴结单独受累和单个结外部位受累组均未达到,多个结外部位受累56.0个月(P=0.273)。尽管三组在PFS和OS未显示出统计学差异,但多结外部位受累患者显示显著的预后不良。244例DLBCL患者中结外受累涉及17个部位,其中肠道38例(38/244,15.6%)、胃34例(34/244,13.9%)、骨骼30例(30/244,12.3%)、骨髓17例(17/244,7.0%)、浆膜(14/244,5.7%)、乳腺12例(12/244,4.9%)、肝脏(11/244,4.5%)、中枢神经系统8例(8/244,3.3%)、甲状腺(7/244,2.9%)、女性生殖系统(7/244,2.9%)、唾液腺(7/244,2.9%)、肺(7/244,2.9%)、睾丸6例(6/244,2.5%)、肾上腺6例(6/244,2.5%)、胰腺(6/244,2.5%)、男性泌尿系统(5/244,2.0%)、皮肤肌肉组织(4/244,1.6%);骨、骨髓、女性生殖系统、浆膜、肝脏、肾上腺、肺、皮肤肌肉中,更容易出现多结外部位受累。2、244例DLBCL患者均在初始治疗前成功检测32个热点驱动基因突变,所有基因均发生了不同频率的突变,其中233例(233/244,95.5%)发生了基因突变,中位突变数5.00(0-24)个,突变频率≥10%的基因有14个,分别是PCLO(38.5%)、PIMI(32.4%)、MLL/MLL2(28.3%)、TP53(27.0%)、CD79B(24.6%)、MYD88(20.5%)、SOCS1(19.3%)、CREBBP(15.2%)、TNFAIP3(14.8%)、CARD11(13.9%)、B2M(11.9%)、IRF4(11.5%)、CCND3(11.1%)、PRDM1(11.1%)。3、分析244例DLBCL患者不同结外部位累及数目与临床病理特征和驱动基因异常的相关性。单因素分析结果显示:无结外部位累及、单结外部位累及和多结外部位累及三组在LDH水平(LDH升高比例:40.7%vs 31.4%vs 68.9%;χ~2=18.938,P=0.000)、Ann Arbor分期(III/IV期比例:42.0%vs 33.9%vs 100%;χ~2=59.212,P=0.000)、IPI评分(3-5分比例:14.8%vs 8.5%vs 88.9%;χ~2=118.326,P=0.000)间的差异具有统计学意义,在年龄、性别、ECOG评分、Hans分型、双表达、TP53蛋白表达间差异无统计学意义(P>0.05),在EZH2(3.7%vs 1.7%vs 15.6%,P=0.002)、MEF2B(6.2%vs 5.1%vs 17.8%,P=0.021)、STAT6(12.3%vs 4.2%vs 2.2%,P=0.033)突变间差异具有统计学意义,在其他29个驱动基因间无差异。多因素分析结果显示IPI评分高、EZH2突变是多结外部位累及的危险因素。4、分析244例DLBCL患者中常见受累部位、特殊部位(胃、肠道、骨、乳腺、中枢、睾丸、肾上腺、骨髓)与驱动基因的相关性,研究发现胃与STAT3突变(OR=6.741,P=0.009)相关;肠道与CD79A突变(OR=9.412,P=0.049)相关;乳腺(OR=4.062,P=0.029)、中枢(n=8;OR=6.771,P=0.014)、睾丸(n=6;OR=20.312,P=0.008)、肾上腺(n=6,OR=8.125,P=0.021)均与MYD88突变相关,其中中枢同时与MYD88 L265P(OR=7.100,P=0.012)、PIM1(OR=6.000,P=0.035)突变相关,睾丸受累同时与MYD88 L265P(OR=7.100,P=0.027)突变相关;骨和骨髓未发现相关驱动基因。结论:1、DLBCL患者中结外受累常见,多个结外部位累及与EZH2基因突变呈正相关。2、DLBCL患者常见的预后不良结外受累部位具有不同的基因突变特征,乳腺、中枢、睾丸和肾上腺与MYD88突变相关,胃、肠道、骨骼和骨髓与MYD88突变无关,其中中枢和睾丸存在MYD88 L265P的热点突变。更多还原

【Abstract】 Background and ObjectiveDiffuse large B-cell lymphoma(DLBCL)is a malignant tumor derived from mature B lymphocytes.It is the most common type of non-Hodgkin’s lymphoma(NHL),accounting for 30% to 40% of adult NHL.DLBCL can occur in almost any part of the human body,including lymphoid organs(Waldeyer’ s ring,spleen,thymus and lymph nodes)and mucosa-associated lymphoid tissue.The common extranodal sites involved are the nasal cavity、sinus、soft palate、salivary gland and thyroid of the head and neck,breast、lung and pleura of the chest,stomach、intestine、liver、pancreas、kidney and adrenal gland of the abdomen,uterus、ovary and testicle of the pelvic,brain、meninges and spinal cord of the central nervous system,skin and soft tissue,bone and bone marrow and so on.International Prognostic Index(IPI)score is the most commonly used tool to evaluate the prognosis of patients with DLBCL,including age,stage,number of extranodal lesions,LDH level and ECOG behavior status.The number of multiple extranodal sites involved(≥ 2)is considered to be one of the poor prognostic indicators.More and more studies have shown that the specific site of involvement is also related to the prognosis.In the National Comprehensive Cancer Network(NCCN)IPI score,central nervous system、bone marrow、liver、gastrointestinal tract and lung involvement are considered to be adverse prognostic factors.Our previous study also found that the prognosis of Waldeyer’ s ring involved was better,while that of central nervous system,bone marrow,breast and adrenal gland were poor.However,poor prognosis of the molecular mechanism and potential accurate treatment of multiple extranodal involvement and different extranodal sites involved are not clear.Due to the high heterogeneity of DLBCL,the existing clinical、morphological and immunophenotype still can not meet the needs of clinical accurate diagnosis and treatment.With the development of high-throughput second-generation sequencing technology,some genes have been found to occur frequently in lymphoma,suggesting that these mutated genes may be involved in the occurrence and development of lymphoma and may be used in the diagnosis,classification and prognosis of lymphoma,and even to guide clinical treatment decisions.In this study,through the detection and analysis of lymphoma-related driver gene abnormalities,we preliminarily explored the correlation between extranodal involvement and lymphoma driver gene abnormalities in DLBCL patients,aiming to reveal the poor prognosis of the molecular mechanism and potential precision treatment of multiple extranodal involvement and different extranodal sites involved.Materials and MethodsThe tumor tissue samples of 244 patients with diffuse large B-cell lymphoma treated in our hospital of from April 2006 to September 2020 were collected.32 hot spot driving gene mutations were detected by the second generation sequencing method.The data were processed and analyzed by SPSS 22.0.,the counting data were expressed by frequency(%).Bilateral χ2 test or Fisher exact probability method was selected for inter-group analysis,and Phi coefficient was used to represent the intensity of association.Logistic regression method was used in multivariate analysis.Kaplan-Meier method was used to analyze the survival,and the survival curve was drawn and Log-rank test was performed.Statistical significance was defined as P <0.05.Results1、Among the 244 patients with DLBCL,extranodal involvement occurred in163 cases,accounting for 66.8% of the total(163/244).Among them,81 cases had no extranodal involvement(81/244,33.2%),118 cases had single extranodal involvement(118/244,18.4%),and 45 cases had multiple extranodal sites(≥ 2)(45/ 244,18.4%).The median follow-up time was 20.4 months.The median progression-free survival time(PFS)was 27.0 months in patients with no extranodal involvement,53.6 months in patients with single extranodal involvement,and 15.6 months in patients with multiple extranodal sites involvement.The median overall survival time(OS)was not reached in the groups with no extranodal involvement and single extranodal site involvement,and multiple extranodal sites were involved for 56.0 months(P=0.273).Although there was no statistical difference in PFS and OS among the three groups,patients with multiple extranodal involvement showed a significantly poor prognosis.There are 17 sites involved in extranodal involvement in 244 patients with DLBCL.Among them,there were 38 cases of intestine(38/244,15.6%),34 cases of stomach(34/244,13.9%),30 cases of bone(30/244,12.3%),17 cases of bone marrow(17/244,7.0%),14 cases of serosa(14/244,5.7%),,12 cases of breast(12/244,4.9%),11 cases of liver(11/244,4.5%),8 cases of central nervous system(8/244,3.3%),7cases of thyroid(7/244,2.9%),and 7 cases of female reproductive system(7/244,2.9%),7 cases of salivary gland(7/244,2.9%),7 cases of lung(7/244.2.9%),6 cases of testic(6/244,2.5%),6 cases of adrenal gland(6/244,2.5%),6 cases of pancreas(6/244,2.5%),5 cases of male urinary system(5/244,2.0%),4 cases of skin and muscle tissue(4/244,1.6%).Multiple extranodal sites are more likely to be involved in bone,bone marrow,female reproductive system,serosa,liver,adrenal gland,lung,skin and muscle.2.32 hot spot driving gene mutations were successfully detected in 244 patients with DLBCL,and all genes had mutations at different frequencies.Among them,233cases(233/244,95.5%)had gene mutations,the median number of mutations was 5.00(0-24).14 genes had mutation frequencies ≥ 10%,they are PCLO(38.5%),PIM1(32.4%),MLL/MLL2(28.3%),TP53(27.0%),CD79B(24.6%),MYD88(20.5%),SOCS1(19.3%),CREBBP(15.2%),TNFAIP3(14.8%),CARD11(13.9%),B2M(11.9%),IRF4(11.5%),CCND3(11.1%),PRDM1(11.1%).3.Analyze the correlation between the number of different extranodal sites involved with clinicopathological features and driver genes abnormality in 244 DLBCL patients.Univariate analysis showed that the three groups of no extranodal site involvement,single extranodal site involvement and multiple extranodal sites involvement at LDH level(LDH elevated ratio: 40.7% vs 31.4% vs 68.9%;χ2 =18.938,P= 0.000),Ann Arbor stage(III/IV stage ratio: 42.0% vs 30.9% vs 100%;χ2= 59.212,P=0.000),IPI score(3-5 score ratio: 14.8 vs 8.5% vs 88.9%;χ2=118.326,P=0.000).There was no significant differences in age,sex,ECOG score,Hans type,double expression and TP53 protein expression.There was significant differences in EZH2(3.7% vs 1.7% vs 15.6%,P=0.002),MEF2B(6.2% vs 5.1% vs17.8%,P= 0.021),STAT6(12.3% vs 4.2% vs 2.2%,P=0.033)and there was no difference among the other 29 driving genes.Multivariate analysis showed that high IPI score and EZH2 mutation were risk factors for multiple extranodal sites involvement.4.The correlation between common involved sites(stomach,intestine,bone,breast,central nervous system,testis,adrenal gland,bone marrow)and driving genes in 244 patients with DLBCL was analyzed.It was found that stomach was associated with STAT3 mutation(OR=6.741,P=0.009);intestine was associated with CD79 A mutation(OR=9.412,P=0.049);breast(OR=4.062,P=0.029)、center nervous system(n=8;OR=6.771,P=0.014)、testis(n=6;OR=20.312,P=0.008),adrenal gland(n=6,OR=8.125,P=0.021)were all associated with MYD88 mutation,in which central nervous system was associated with MYD88 L265P(OR=7.100,P=0.012)and PIM1(OR=6.000,P=0.035)mutation,testis involvement was also associated with MYD88L265P(OR=7.100,P=0.027)mutation;no related driving genes were found in bone and bone marrow.Conclusions1.Extranodal involvement is common in DLBCL patients,and multiple extranodal involvement is positively correlated with EZH2 mutation.2.The common extranodal sites with poor prognosis in DLBCL patients have different gene mutation characteristics.Breast,CNS,testis and adrenal gland are associated with MYD88 gene mutation,while stomach,intestine,bone and bone marrow are not associated with MYD88 mutation,among which there are hot spot mutation of MYD88 L265 P in CNS and testis.更多还原