博士学位论文伴t(8;21) ( q22;q22) 急性髓系白血病微小残留病监测及意义学校代码:10023学号:B2012014018K562细胞中HtrA2对WTl 的调控意义及机制探讨所院:血液病医院( 血液学研究所)姓名:张丽霞指导教师:秘营昌教授导师小组:王建祥王敏饶青教授学科专业:内科学( 血液病学)研究方向:白血病临床与基础完成时间:2014年4月 中国医学科学院北京协和医学院博士学位论文目录第一部分伴t( 8;21) ( q22;q22) l 拘急性髓系白血病微小残留病监测及意义⋯ ⋯ .1中文摘要⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯⋯.3英文摘要⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ..4前言⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ .6一日U舌⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ .材料与方法⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ .7结果⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ..15讨论⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ 一⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ..23参考文献⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ 26第二部分K562细胞系中HtrA2对WTl 的调控意义及机制探讨⋯ ⋯ ⋯ ⋯ ⋯ .29中文摘要⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ 31英文摘要⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ .32前言⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ 3月U舌⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ..j 4材料与方法⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ .36结果⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ .48讨论⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ..6l参考文献⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ .65综j 苤⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ .66攻读学位期间发表的文章题录⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ 74缩略词表⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ 75致谢⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ 76独创性声明⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ ⋯ 77 中国医学科学院北京协和医学院博士学位论文第一部分伴t( 8;21) ( q22;q22) 急性髓系白血病微小残留病监测及意义1 中国医学科学院北京协和医学院博士学位论文2 中国医学科学院北京协和医学院博士学位论文摘要研究目的:分析急性髓系白血病伴t(8;21)(q22;q22)患者初诊、治疗及随访过程中融合基因AMLl /ETO定性及定量监测的变化规律及预后意义。研究方法:总结分析52例急性髓系白血病伴t( 8;21) ( q22;q22) 患者初诊、治疗及随访过程中融合基因—wLl /ETO定性检测(32例)转阴情况及定量检测(20例)表达水平变化规律;统计分析融合基因转阴情况及表达变化趋势与患者复发及生存情况的关系。结果:32例AML伴t( 8;21) ( q22;q22) 患者在各时间点/区间( 完全缓解,即CR时,CR后0.3月,3-6月,6.12月,12.18月,18.24月,>24月) 融合基因AMLl /ETO转阴率分别为20%,46.88%,71.88%,71.88%,72.41%,95.45%,100%。不同时间点融合基因阳性/阴性组患者复发情况:CR时( 复发例数/总例数) 为3/20 vs.3/5,P=0.07;CR后0.3月为3/17 VS.3/15,P=I.0;CR后3-6月为2/9 VS.4/23,P=I.0;CR后6.12月为5/9 VS.1/23,P=0.003;CR后12.18月为5/8 VS.0/21,P=O.000:CR后1 8.24月为0/1 VS.0/21;CR后>24月时17例均阴性,0/17。生存分析显示:CR时、CR后6.12月、12.18月融合基因定性结果与生存相关,3年无复发生存( RFS) 分别为85%VS40%,P=0.037;44.4%VS 95.5%,P=0.000;37.5%VS 100%,P=0.000:3年总生存( OS) 分别为81.4%VS 40%,P=O.01 8;25%VS 95.7%P=0.001;20%VS 100%P=0.000。融合基因定量分析显示,初诊时患者融合基因拷贝数与复发无关。CR时融合基因下降程度( <2对数级和≥2对数级) 与RFS相关,1年RFS为40%VS 100%,P=0.02。CR后3个月,5/6个月时融合基因拷贝数与复发相关:CR后3个月时,复发组VS.无复发组拷贝数分别为300 VS 10.5拷贝/104ABL基因,P=0.039;在CR后5/6个月时,两组中位拷贝数分别为2161vs 12拷贝/104ABL基因,P=0.004。缓解后治疗过程中融合基因定量的升高( ≥O.5对数级) 同样影响RFS,1年RFS分别为58.3%VS 100%,P=0.017。结论:融合基因AMLl /ETO定性、定量监测均可预示患者预后。CR后6个月后融合基因转阴性的患者长生存的几率较大;CR时融合基因定量下降幅度大、监测过程中持续低水平的患者长生存的机会较大。关键词:微小残留病;急性髓系白血病;t( 8;21) ( q22;q22) ;AMLl /ETO:PCR 中国医学科学院北京协和医学院博士学位论文Detecti on of Mi ni mal Resi dual Di seasei n AcuteMyel oi dLeukemi a wi tht(8;21)(q22;q22)AbstractObj ecti ve:Toeval uate theprognosti c i mpact of AMLl /ETO fusi ongenedetecti onqual i tati vel yandquanti ti vel yi npati entswi thacutemyel oi dl eukemi a、衍th“ 8;21)(q22;q22)[t(8;21)一AML].Methods:The resul ts of AMLl /ETOfusi ongenedetecti on wereanal yzedf rom52t( 8;21) -AML pati ents(qual i tati ve anal ysi sof 32pati entsandquanti ti ve anal ysi sof 20pati ents)duri ngthe ti me of fi rstdi agnosi s,therapeuti c processandfol l ow—up,tofendtherel ati onshi pbetween thechangeof AML1/ETO fusi ongene expressi onand thepati ents’ rel apseand survi val .Resul ts:Thenegati verate ofAMLl /ETOf rom32pati entsat our checkpoi nt/regi ons(getti ng compl ete remi ssi on,0-3,3-6,6—12,12-18,18-24 and>24months f romCR)wereas fol l ows:20%,46.88%,71.88%,71.88%,72.41%,95.45%and100%,respecti vel y.The rel apseofpati ents f romdi fferent groups( AML1/ETOposi ti veornegati ve) atevery checkpoi nt/regi onwasas bel l ow:whengetti ng CR( rel apsi ngpati ents/al l pati entsof thegroup),3/20 VS.3/5,P=0.07;0-3 months f romCR,3/17VS.3/15,P=I.O;3-6months f romCR,2/9 VS.4/23,P=-I.O;6-12 months f romCR,5/9VS.1/23,P=0.003;12—18 months fromCR,5/8VS.0/21,P=0.000;18- 24 months fromCR0/1VS.0/21;>24 months f romCR,al l1 7pati ents werenegati ve,0/I7.Survi valanal ysi sshowed that the AML 1/ETOresul ts of these three checkpoi nt/regi ons(whengetti ng CR,6- 12,12-18 months f romCR) were rel ated to survi val .Threeyearsrel apsefl ee survi val ( RFS) were 85%VS 40%,P=0.037;44.4%VS 95.5%,P=0.000;3 7.5%VS 100%,P=0.000;3 yearsoveral l survi val ( OS) were respecti vel y:81.4%VS40%.P=O.018;25%VS 95.7%P=0.001;20%VS 100%P=0.000.Quanti tati ve anal ysi sof AML1/ETO fusi ongeneshowed that there was norel ati onshi pbetween the i ni ti alcopynumberatdi agonosi s andrel apse rate.Addi ti onaldata refl ected thatthedecl i ni ng degree of AML 1/ETOcopi es whengetti ng CR( <2 l ogor≥2l og)i nfl uenced RFS;lyearRFS was 40%VS100%.P=0.02.Thecopynumberat 3months and 5/6 months after CR were rel ated torel apse:at3 months after CR.thecopy numberwas300 VS.10.5copi es/10斗ABLi nrel apsed and non—rel apsedgroup(P=0.039).and2161 VS.12copi es/104ABL at5/6 months aRerCR(P=0.004).4 中国医学科学院北京协和医学院博士学位论文Andri si ngof AMLl /ETOcopi esafterCR(≥0.5 l og)al soaffected RFS;1 yearRFSwas 58.3%VS 100%.P=O.01 7.Concl usi on:Qual i tati vel y moni tori ng AML1/ETO fusi on genecanpredi cttheprognosi sof t(8;21) 一AML pati ents.Pati entsachi evenegati veresul ts after 6 monthesfromCRmayhavel ongersurvi val ti me.Inquanti tati ve detecti on,the l argerdecl i neddegreeof AML1/ETOcopi esat the ti me of getti ngCR andconstantl y keepi ngl owl evelduri ng moni tori ngi ndi cates better survi val .Keywords:Mi ni mal resi dual di sease,acutemyel oi dl eukemi a, t( 8;21)(q22;q22)AMLl /ETO.PCR5 中国医学科学院北京协和医学院博士学位论文—1上.—j L·日!