evi1基因在儿童白血病的表达及临床意义

1、evi1基因在儿童白血病的表达及临床意义博士研究生姜敏博士生导师金润铭教授华中科技大学同济医学院附属协和医院儿科中文摘要白血病是儿童时期最常见的恶性肿瘤性疾病，是造血组织中某一类型的白血病细 胞在骨髓或其他造血组织中恶性增生，并浸润体内各脏器、组织，导致正常造血细胞 受抑制，产生各种症状。近十年来，用基因方法研究儿童白血病取得了巨大突破，我们对白血病的认识更 加深入。基因检测有助于我们探究白血病的病理发生机制、指导危险分度、预后评估 及制定相应治疗策略，从而大大提高了儿童白血病的生存率。根据基因研究确定新的 治疗靶点，进行个体化的靶向治疗也是目前片血病治疗发展的方向。人类evi1 (ecotr

2、opic viral integration site)基因是定位在染色体的3q26位点， 编码一个锌指转录因子，定位于核，在rna的转录调节中发挥作用。其异常表达的机 制包括3q重排、7号染色单体、mll易位或与其它基因相互作用。研究发现evt1基 因在部分急性淋巴细胞性口血病、急性髓细胞性口血病和慢性粒细胞性口血病屮都有 异常表达，并且与不良预后相关，但其预后价值尚存争议，致口血病机制也不明确。本研究通过分子生物学技术，如逆转录一聚合酶链反应法、实时定量聚合酶链反 应法、流式细胞术、染色体检查等并结合患儿的临床资料，探讨evi1基因在儿童口 血病的表达情况及生物学本质。第一部分evi1基因

3、在儿童急性淋巴细胞性白血病中的表达及临床意义目的：研究evi1基因在儿童急性淋巴细胞性白血病的表达情况，evi1基因的生 物学特征及临床意义，方法：收集262例2010年12月到2013年2月华中科技大学同济医学院附屈协 和医院儿科初诊all患儿的临床资料，采用巢式逆转录-聚合酶链式反应法、实时定 量聚合酶链反应法、流式细胞术、染色体等检测evi1基因表达的临床特征及生物学 特征，结果：262例急性淋巴细胞性白血病患儿，共有29例evi1基因表达为阳性，阳性 表达率 11. 07%o 包括 27 例 b 系 all (93. 10%)和 2 例 t 系 all (6. 90%)。evi1 基因

4、 在all不同危险度亚组的表达率比较，差异有统计学意义，高危组与标危组比较，高 危组的evi1基因的阳性表达率明显提高，差异有统计学意义(p0. 05)o evi1基因表 达阳性all患儿女性比例增加，初诊时外周血白血病计数升高，血小板计数降低，差 异有统计学意义(p0.05),而血红蛋白含量和年龄两组间比较，差异无统计学意义 (p>0. 05)。免疫分型检测示b系evi1基因表达阳性组all患儿高表达ccd79a( 100%)、 cd22(81.48%)、cd19(8148%)、cd10(81.48%)、cd38(81.48% )、tdt(81.48%)、 cd123(81. 48%)

5、、hladr(77. 78%)以及 cd34(70. 37%), b 系 all 中 evi1 基因表达阳性 患儿髓系相关抗原(cd13、cd33、cdllb)表达明显增加，b淋巴细胞相关抗原(cd19、 cd20、cd22)表达明显减少，差异有统计学意义(p0. 05)。t系evi1基因表达阳性 组的 all 患儿高表达 ccd3 (100%)、cd38 (100%)、cd8 (100%)、cd7 (100%)、cd5 (100%)、 cd4 (100%) cd3 (100%)、cd2 (100%)。部分all患儿evi1基因表达改变与临床缓解 不同步,与mrd改变也不同步。20. 69%(

6、6/29) evi1基因表达阳性的all患儿检测到 其它融合基因共表达。核型分析示evi1基因表达阳性的all患儿核型可正常，25%(6/24)患者有染色体结构或数目的异常，包括3q位点异常、11号染色体重排、7 号染色单体等。29例evt1基因表达阳性的all患儿中有68. 97% (20/29)患儿强的松 试验敏感，第一疗程cr率为57. 69% (15/26)；总cr率65. 38% (17/26),总死亡率为13. 64% (3/22),复发率为9. 09% (2/22)。evi1基因表达阳性组与evi1基因表达阴 性组相比，evi1基因表达阳性组强的松试验敏感者明显减少，第一疗程cr

7、率和总cr 率明显降低，差异有统计学意义(p0.05),而总死亡率和复发率比较，差异无统计 学意义(p>0. 05)o随访时间截止至2013年2月，对22例evi1表达阳性和176例evi1 表达阴性的all患儿采用kaplan-meier生存分析法进行分析。ev11表达阳性组1年、 2年累计efs率分别为(58. 9±10. 6) %, (50. 0±10. 7) %, ev11表达阴性组1年、2 年累计 efs 率(88.2±5.4) %、(85. 2±5. 6) %,通log-rank 检验，evi1 表达阳性 组的1年、2年累计efs率比e

8、vi1表达阴性组明显降低，差异有统计学意义(p0. 05)o结论：在儿童al±中ev11基因可异常表达，evi1基因表达阳性患儿预后差。关键 词：ev11基因；急性淋巴细胞性白血病；儿童第二部分evi1基因在儿童急性髓细胞性白血病中的表达及临床意义目的：研究ev11基因在儿童急性髓细胞性白血病的表达情况、ev11基因的生物 学 特征及临床意义，方法：收集241例2009年1月至2013年8月华中科技大学同济医学院附屈协和 医院儿科初诊aml患儿的临床资料，采用巢式逆转录-聚合酶链式反应法、实时荧光 定量聚合酶链反应法、流式细胞术、染色体检查等检测evi1基因表达的临床特征及 生物学特

9、征，结果：241例aml患儿中，有33例ev11基因表达呈阳性(13.69%)；其中低危0 例(0%),中危7例(21.21%),高危26例(78. 79%),包括10例m2型，11例m4型, 5例m5型,6例m6型,1例m7型;而mo、ml、m3型无evi1基因阳性表达。evi1 基因在各aml亚型的的阳性表达率明显不同，差界有统计学意义(p0.05)。ev11基 因在aml不同危险度亚组的表达率有差异，evi1基因在高危aml患儿屮的阳性表达 率比低危和中危aml患儿明显提高(p0.05)。evi1阳性的aml患儿初诊吋女性患儿 比例增加(p<0. 05),而初诊年龄、外周血wbc、

10、hb含量、plt计数、肝脾肿大和淋巴 结肿大的发生率比较，差异无统计学意义(p>005)。evi1基因阳性的aml高表达 cd33(100%),cd38(87. 88%), hladr(75. 76%)。并且cd64和cd71 的表达明显升高,cd123 的表达明显降低，差异有统计学意义(p<0. 05)o部分evi1基因表达的改变与临床缓 解、mrd改变不同步。39. 39% (13/33)的evi1基因表达阳性aml患儿中检测到有其 它融合基因共表达。核型分析示50% (15/30)核型正常；50% (15/30)患者有染色体 结构或数目的异常。包括3q位点异常、11号染色体重

11、排，7号染色单体等。33例evi1 基因表达为阳性的aml患儿第一疗程cr率为23%(7/31)；总cr率为45. 16%( 14/31), 总死亡率为60. 71% (17/28),复发率为10. 71% (3/28)。aml患儿evi1表达阳性组与 evi1表达阴性组相比,evi1表达阳性组第一疗程cr率、总cr率明显降低，总死亡 率明显升高，差异有统计学意义(p0.05),而复发率比较，差异无统计学意义 (p>0. 05)o随访时间截止至2013年8月，对28例evt1表达阳性和165例evt1表达阴性的aml患儿采用kaplan-meier生存分析法进行分析。evi1基因表达阳性

12、组1 年、4年累积efs分别为(34. 0±9. 2) %、(21. 3±9. 3) %, evi1基因表达阴性组1 年、4年累积efs分别为(65.3±3.9) %、(51.5±3.7)，通过log-rank检验，两组 间差异有统计学意义(p0.05)。结论：aml患儿+ evi1基因表达阳性提示不良预后；在aml的病程屮evi1基因的活化不是孤立的，而是与其它基因相互作用，其机制及功能还需要进一步研究。关键词：evi1基因；急性髓细胞性口血病；儿童第三部分evi1基因在儿童慢性粒细胞性白血病中的表达及临床意义目的：研究evi1基因在儿童慢性粒细胞性白

13、血病的表达情况及临床意义。方法: 收集并分析11例2009年1月至2013年8月华中科技大学同济医学院附屈协和医院儿科血液病病区初诊的cml患儿的临床资料，采用巢式逆转录-聚合酶链 式反应法、流式细胞术、染色体检查等检测ev11基因表达阳性患儿的临床特征及生 物学特征，结果:11例cml患儿初诊时均检测到bcr/abl融合基因，其中有3例evi1基因表 达阳性，阳性表达率为27. 27%,1例为cml加速期,2例为cml慢性期,ev11基因表达阳性与表达阴性的cml患儿初诊时的临床特征相比，年龄、性别、外周血wbc、 hb含量、plt计数、嗜酸性粒细胞绝对值、嗜碱性粒细胞绝对值、肝脾肿大及淋巴

14、结 肿大发牛率的差异均无统计学意义（p>0.05）。evi1基因表达阳性的cml患儿中可见 cd33、cd38高表达。经伊马替尼治疗后，3例evi1基因表达阳性患儿中有2例bcr/abl 基因和evt1基因均未转阴，而1例cml患儿bcr/abl基因未转阴，evt1基因已转阴。 3例evi1基因表达阳性cml患儿有2例分别在治疗后1月、3月达到血液学缓解，完 全缓解率为66. 67%（2/3）,与evi1基因表达阴性组相比，差异无统计学意义（p005）。3 例evi1基因表达阳性cm患儿均存活，无复发。其总生存期（0s）分别为14、13、 20月，无事故生存期（efs）分别为14、13、

15、0月。结论：evi1表达阳性的cml是一种特殊类型的白血病，其临床特征无特异性，预 后价值还需进一步明确。临床上需动态监测evi1基因表达，了解这种特异的基因表达 信号的生物学及临床重要性。关键词：evi1基因；慢性粒细胞性白血病;儿童expression and clinical significanceof evi1 gene in childhoodleukemiadoctor candidate: jiang minsupervisor: prof. jin runmingpediatrics department of union hospital, tongji medical c

16、ollegehuazhong university of science & technologyabstractleukemia is the most common malignant diseases of childhood in which malignant hyperplasia of a type of leukemic cells are found in the bone marrow hematopoietic tissue or other malignant hyperplasia, infiltrating organs and tissues, resul

17、ting in inhibition of normal hematopoietic cells and producing a variety of symptoms.in the past ten years, the study of childhood leukemia by gene method has made so great breakthrough that we can understand leukemia more deeply.genetic testing help us to explore the pathogenesis of leukemia, guide

18、 risk stratification, prognosis evaluation , make treatment strategies,which greatly improve the survival rate of childhood leukemia. at present to identify new therapeutic targets according to the genetic research and give targeted therapy for individual is the direction for leukemia treatment.the

19、human evi1 ( ecotropic viral integration site) gene is located in chromosome 3q26 locus , encodes a zinc finger transcription factor and play arole in rna transcriptional regulation. evi1 gene is located in the nucleus.the abnormal expression mechanisminclude 3q rearrangement, monosomy 7,mll translo

20、cations or reaction with other genes. studies have shown that evi1 gene can aberrant expressed in acute lymphatic leukemia, acute myeloid leukemia and chronic granulocytic leukemia . the expression of evi1 gene is correlated with poor prognosis but its prognostic value remains controversial and leuk

21、emogenic mechanism is not clear.in the study we investigated the expression of evi1 gene in childhood leukemia and biological characteristic by molecular biology techniques such as nested reverse transcription-polymerase chain reaction , real time quantitative polymerase chain reaction , flow cytome

22、try, karyotypic detection and clinical data.part i expression and clinical significance of evi1 gene inchildhood acute lymphocytic leukemiaobjective : to study the expression 、 clinical significance and biological characteristics of ecotropic viral integration site ( evi1 ) gene in childhood acute l

23、ymphoblastic leukemia(all).methods: the clinical data from 262 newly diagnosed all children in department of pediatrics, union hospital, tongji medicalcollege,huazhonguniversity of scinence and technology were obtained and analyzed. fromdecember, 2010 to february , 2013 、 nested reverse transcriptio

24、n- polymerase chain reaction , real time quantitative polymerase chain reaction , flow cytometry, karyotypic detection were used for detection the expression of evi1 gene and the clinical and biological characteristics.results : 29 cases with evi1 gene positive were found among 262 childhood all,inc

25、luding 27 b-all (93.10%) and 2 t-all (6.90%) . the incidences of positive expression was 11.07%. there were signficant difference among different risk groups of all respect to evi1 gene expression rate. the positive expression rate of evi1 gene increased significantly in the high risk group than sta

26、ndard risk group, (p<0.05). compared with evi1 gene negative expression group，all with evi1 positive had more female children , higher white blood cell count and lower platelet count (p<005 ) but there was no signficant difference about age and hemoglobin (p>0.05) . ccd79a (100% ) > cd22

27、(81.48%)、cd 19(81.48%) > cd10(81.48%) > cd38(81.48% )、 tdt(8l48%)、cd 123(81.48%) > hladr(77.78%) and cd34(70.37%) were highly expressed in b-all with evi1 gene positive . the expression of parts of myeloid lineage-associated antigens (cd13, cd33, cdllb) were significantly increased and part

28、s of b lymphiod lineage-associated antigens (cd 19, cd20, cd22) were significantly decreased, in childhood all with evi1 gene positive (p<0.05) . ccd3 (100%)、cd38(100%)、cds (100%)、cd7 (100%)> cd5 (100%)、cd4 (100%) cd3 (100%)、 cd2 (100%) were highly expressed in t-all with evi1 gene positive. t

29、he clinical remission and changes of evi1 gene expression were not synchronized for parts of all childhood, and the mrd changes were not synchronous also. there were 20.69 % (6/29) of all with evi1 gene positive coexpressed with other fusion genes. besides normal karyotype , abnormal chromosome stru

30、cture or number were found in 25% (6/24) of childhood with evi1 positive .including 3q locus abnormal, chromosome 11 rearrangement, monosomy 7 and so on. 20 (68.97%) cases of childhood with evi1 gene positive had prednisone good-response 26 cases completed remission induction therapy the first cours

31、e of treatment cr rate was 57.69% (15/26) and the total cr rate was 65.38% (17/26),.total mortality rate was 13.64% (3/22).the recuitence rate was 9.09% (2/22).the number of prednisone good-response was significantly decreased and complete remission rate of the first and total courses of treatment w

32、as significantly decreased (p<0.05), but total mortality rate and recurrence rate has no obvious difference(p>0.05) in all with evi1 positive childhood comparing with evi1 negative followed up to february, 2013, 22 cases of evi1 positive and 176 cases of evi1 negative were analyzed by kaplan-m

33、eier survival analysis.the 1 year and 2 year cumulative efs were(5&9±106)%, (500±107)% in all with evi1 gene positive,which was significantly lower than (88.2±5.4)%、(85.2±5.6)% in evi1 gene negative by log-rank test( p<0.05).conclusion： the evi1 gene can be abnormal expres

34、sed in childhood all,and the prognosis is poor in childhood all with evi1 gene positive expression.key words： ecotropic viral integration site; acute lymphocytic leukemia;childhoodpart ii expression and clinical significance of evi1 gene in childhood acutemyeloid leukemiaobjective :to study the expr

35、ession , clinical significance and biologicalcharacteristics of ecotropic viral integration site (evi1) gene in childhood acute myeloid leukemia(aml).methods: the clinical data from 241 newly diagnosed aml children in department of pediatrics, union hospital, tongji medical college, huazhong univers

36、ity of scinence and technology were obtained and analyzed. from january, 2009 to august , 2013 , nested reverse transcription-polymerase chain reaction , real time fluorescence quantitative polymerase chain reaction .flow cytometry,karyotypic detection were used for detection the expression of evi1

37、gene and the clinical and biologicalresults： there were ,：33characteristics.cases of evi1 gene positive in 241 childhood amland the incidences of positive expression was 13.69%.among them, 0 case of standard risk, 7 cases of intermediate risk, 26 cases of high risk were included theevi1 gene positiv

38、e expression were found in 10 cases of m2, 11 cases ofm4,5 cases of m5,6 cases of m6,1 cases of m7 , but not in mo、ml、m3 subtypes,there were signficant difference among different risk groups of aml and different subtypes respect to evi1 gene expression rate. the evi1 gene positive expression rate in

39、creased significantly in the high risk group than the other risk groups (p<0.05). compared with evi1 gene negative expression group , aml with evi1 positive at first visit had more female children(p<0.05) .but there were no signficant difference about age， wbc，hb,plt and the rate of hepatosple

40、nomegaly and lymphadenopathy (p>0.05).cd33(100%),cd38(87.88%) and hladr(75.76%) were highly expressed inaml with evi1 gene positive .the expression of cd64 and cd71 in aml with evi1 gene positive were significantly higher and cd 123 were significantly lower than aml with evi1 gene negative. (p<

41、;0.05) . the clinical remission and mrd changeswere not synchronized with the changes of evi1 gene expression for parts of aml childhood , there were 39.39%(13/33) of aml with evi1 gene positive coexpressed with other fusion genes. besides normal karyotype , abnormal chromosome structure or number w

42、ere found in 50% (15/30 ) of childhood with evi1 positive by karyotype analysis .including 3q locus abnormal, chromosome 11 rearrangement, monosomy 7. 31 of 33 cases with evi1 positive completed remission induction therapy . the first course of treatment cr rate was 23% (7/31) and the total rate of

43、cr rate was 45.16% (14/31)total mortality rate was 60.71% (17/28) .the recurrence rate was 10.71% (3/28) . the complete remission (cr) rate of the first and total course of treatment were significantly decreased and the total mortality rate were significantly increased (p<0.05 ) ,but the recurren

44、ce rate had no obviousdiflcrcncc(p>0.05) in aml with evi1 positive childhood comparing with evi1 negative followed up tg#ugust, 2013,and 165 cases of evi1negative were analyzed by kaplan-meier survival analysis. the 1 year and 4 year cumulative efs were (34.0±9.2)% ,(21.3±9.3)% in aml w

45、ith evi1 gene positive,which was significantly lower than (65.3±3.9)%,( 51.5±37) in evi1 gene negative by log-rank test (p<0.05).conclusion： the positive expression of evi1 gene in aml point out poor prog nosis .in the development of aml，the activation of evi1 gene is not isolated but t

46、he results of interactions with other genes the mechanism of activation and function of evi1 gene needs further study.key vvords:ecotropic viral integration site; acute myeloid leukemia;childhoodpart iii expression and clinical significance of evi1 gene in childhood chronic myelogenous leukemiaobjec

47、tive： to study the expression , clinical significance of ecotropic viral integration site (evi1) gene in childhood chronic myelogenous leukemia (cml).methods: the clinical data from 11 newly diagnosed cml children in department of pediatrics, union hospital, tongji medical college, huazhong universi

48、ty of scinence and technology were obtained and analyzed from january, 2009 to august , 2013 , nested reverse transcription-polymerase chain reaction ,flow cytometry,karyotypic detection were used for detection the expression of evi1 gene and the clinical and biological characteristics-results： bcr/

49、abl confusion gene was detected in 11 cases of newly diagnosed childhood cml there were 3 cases with ev11 gene positive expression in 11 childhood cml and the incidences of positive expression was 27.27%£ among 3 cases, 1 case was accelerated phase of cml、2 cases were initially diagnosed as the

50、 chronic phase of cml,there were not signficant difference between evi1 gene positive expression group and negative group at first visit respect to clinical manfestication such as age, sex, peripheral wbc, hb content, plt count，eosinophil absolute value, basophils absolute value and the rate of hcpa

51、tosplcnomcgaly and lymphadcnopathy (p>0.05 ). cd33(100%) ,cd38(100%) were highly expressed in cml with evi1 genepositive .after treated with tyrosine kinase inhibitors imatinib, 2 cases of 3 childhood cml with evi1 gene positive didnot acquire conversion of bcr/abl and evi1 gene, while 1 case acq

52、uired conversion of evi1 gene but not bcr/abl gene. in 3 cases childhood cml with evi1 gene positive 2 cases had get hematology complete remission(cr) after 1 month, 3 months treatment and the cr rate was 66.67% (2/3)， which was not differencecompared with evi1 gene negative group50.00%(4/8) (p>0

53、.05) . all of the 3 cases with evi1 positive were survival and no recurrence .the overall survival (os) were 14、13> 20 months and disease free survival(efs) were 14 13 0 months respectively.conclusion： cml with evi1 gene positive expression is a relatively rare subtype of leukemia , which have no

54、t signficant difference respect to clinical manfcstication and its prognosis significance is not clear.dynamic monitoring theexpression andexploring the biological and clinicalimportance about thedistinctive geneexpression signal are needed key words： ecotropic viral integration site; chronic myelog

55、enous leukemia ；childhoodzhi ku quan 20150721第一部分evi1基因在儿童急性淋巴细胞性白血病中的表达及临床意义月us急性淋巴细胞性白血病(acute lymphocytic leukemia, all)是小儿最常见的 恶性肿瘤性疾病，大约70%-85%的儿童白血病为all。all根据白血病细胞恶变的细胞 系列分为t系all和b系all,其中b系all占绝大多数。目前all的治疗主要是根据初诊时年龄和白细胞计数等临床特征进行危险分层，进一步根据micm分型,即骨髓细胞形态学(morphology, m),免疫学(immunology,i),细胞遗传学(

56、cytogenetics, c),分子生物学(molecularbiology, m)以及早期治疗反应、微小残留病等进行危险分层，追求all的早期诊断和个体化治疗。对低危患儿要求进场也度 1 化疗畑免化o#飙据副1 对3丿21行强化治疗，避免耐药和复发，这种危险分层和个体化的治疗方案明显提高了 all无事件生存率。但是根据目前的危险分层，很多有高危因素的患儿缺少与不良预后有关的细胞遗传 学和分子牛物学改变，而j4尽管用了更强的化疗，部分高危all患儿预后仍不好；而 临床特征和mtcm分型提示预后良好以及早期治疗反应良好的患者也占了复发和死亡 患者很大一部分比例。因此近年来，白血病研究领域正在积

57、极寻找新的预后相关基因 ,用以指导临床诊治。人类热带病毒整合位点(ccotropic viral intcgration site, evi1)基因是定 位在染色体的3q26位点，发现于1988年，是由mucenski等首先在akxd鼠髓系恶性 肿瘤dna中鉴定出。ev11基因编码一个锌指转录因子，其相对分子质量为14. 5万道 尔顿，这个转录因子定位于核，是位点特异的dna结合蛋白，在rna的转录调节中发 挥作用。根据文献报道，在部分髓细胞性白血病中可以检测到evi1基因的异常表达， 且与不良预后相关0役长期以来evi1基因一直被人们作为一个髓系肿瘤的相关基因 而硏究，前期的文献均报道evi

58、1基因具有髓系特异性，而有关evi1基因在all中的 报道却很少。evi1基因是否在all中表达，表达特征如何，其生物学作用是什么， 能否成为all新的预后因子，能否成为口血病靶向治疗的靶点，尚不清楚。为此，本 研究利用先进的分子生物学技术，如巢式逆转录-聚合酶链式反应(nested reverse transcription-pol)nnerase chain reaction, nested rt-pcr) 法、实时定量 pcr (quantitative real-time polymerase chain reaction, qrt-pcr)、流式细胞术、 染色体分析等检测，并结合患儿

59、的临床资料，研究ev11基因在儿童all的表达特征 及其与预后的相关性，初步探讨evi1基因的生物学本质，为进一步ev11基因致病机 制的研究及靶向治疗的研究打下基础。zhi ku quan 20150721病例与方法1病例收集2010年12月到2013年2月就诊于华屮科技大学同济医学院附属协和医院儿 科all初诊病例共262例，其中男性134例，女性128例；中位年龄为5. 6岁(0. 5 岁-12岁)，经骨髓细胞学和免疫分型检测，所有病例均符合fab诊断标准，都未接受 过化疗。依据evt1基因的表达，将患儿分为阳性组和阴性组。并选取非恶性疾病患 者10例为对照组，男性6例，女性4例；中位年龄为5. 2岁(0.8岁-10岁)。随访 时间截止至2013年2月28日,中位随访时间为18个月(1-26个月)。2方法2. 1收集临床资料收集患儿初諺 4的l卜周血血红蛋白含量(!)、必k2 jfa小板计数(plt)等临床资料。按照中国儿童白血病协作组-08方案对所有患儿进行危险分层和治疗，分为标危 组、中危组以及高危组。危险度标准如下：标危组(sr)：必须同时