【作者】 吴春晓；

【导师】 仇惠英； 潘金兰；

【作者基本信息】 苏州大学 ， 内科学， 2012， 硕士

【摘要】 目的1.系统分析257例自2001年至2011年在本实验室进行常规核型分析检测出20q-异常血液病患者的临床及实验室特征，对比国外报道，揭示我国20q-血液病患者独特的临床、细胞遗传学、预后特点。2.明确20q-衍生异常i(20q-)是否具有独特的骨髓细胞形态学变化特点及预后。3.分析伴20q-异常不同血液肿瘤间是否存在明显差异的缺失区域、共同缺失区域、扩增区域，从而为研究位于异常DNA拷贝数区域内的肿瘤相关基因提供一定信息。4.了解20q-继发t(20;21)(q11;q11)核型异常血液病患者的临床和实验室特征。方法1.257例20q-患者均采用直接法或短期培养法制备染色体，以热变性R显带技术进行核型分析，选取其中有染色体悬液标本保存的196例患者，应用20q11/12双色探针进行FISH验证。按照FAB协作组分型标准对患者进行疾病分类，对其中109例患者进行随访，进行Kaplan-Meier生存分析。2.采用20q12/20qter组合探针对i(20q-)患者进行FISH验证，选择有骨髓片保存的i(20q-)、单纯20q-及20q-伴随其他染色体异常患者进行骨髓细胞形态学回顾性分析，对三组患者进行生存分析。3.从257例患者中筛选出21例有细胞保存的标本（其中1例为i(20q-)），送往上海伯豪生物技术有限公司进行全基因组aCGH检测，以正常人骨髓标本作实验对照。4.对t(20;21)患者采用20q11/12双色探针、20号和21号染色体着丝粒探针、20号和21号染色体亚端粒探针以及20号和21号染色体涂染探针进行荧光原位杂交(FISH)研究。结果1、257例伴20q-异常恶性血液病患者的临床及细胞遗传学特征257例20q-患者中男性163例，女性94例，患者年龄主要集中于50-59岁和60-69岁。其中MPN13例（5.1%）；MDS127例（49.4%）；AML45例（17.5%）；CML5例（2.0%）；ALL15例（5.8%）；AL未分型6例（2.3%）；嗜酸细胞增多症2例（0.8%）；CLL2例（0.8%）；MH1例（0.4%）；巨球蛋白血症1例（0.4%）；诊断未明确40例（15.6%）。257例患者均接受了CC检测，其中单纯20q-120例（46.7%），20q-伴随一种染色体异常32例（12.5%），20q-复杂异常72例(28.0%),i(20q-)28例（10.9%），双20q-5例（1.9%）。各组均为男性多于女性，各组年龄、白细胞水平、血红蛋白水平、血小板水平差异无统计学意义（P＞0.05）。在20q-伴随其他染色体异常组中，其余22条染色体均有涉及，发生频率较高的有5号染色体异常（41%），7号染色体异常（40%）和9号染色体异常（39.7%）。对196例患者进行FISH检测，接近50%的患者FISH阳性信号为一红一绿，表明既有20q12的缺失，也有20q11的缺失，且各组大部分病例20q-阳性细胞比例大于50%，即克隆群体较大。生存分析显示，单纯20q-组中位生存时间（46个月），长于20q-伴随其他染色体异常组（11个月）（P=0.00）；进一步分析得出单纯20q-中位生存时间长于20q-伴随一种染色体异常组（14个月）（P=0.001）；20q-伴随一种染色体异常与20q-复杂异常组中位生存时间(8个月)比较无统计学意义（P＞0.05）；20q-复杂染色体数目与结构异常组中位生存时间（11个月）长于20q-复杂单纯结构异常组（4个月）（P＜0.05），20q-小克隆组中位生存时间长于20q-大克隆组（P＜0.05），阳性信号为2G1R的患者与信号为1G1R的患者中位生存时间比较无统计学意义（P＞0.05）。2、i(20q-)核型异常患者的细胞形态学与预后分析本实验室10年来共有28例伴i(20q-)异常患者，其中男性17例，女性11例，占20q-患者总数的10.9%。i(20q-)组与单纯20q-组相比，骨髓细胞出现少颗粒及空泡化中性粒细胞，少颗粒及空泡形成嗜酸粒细胞，中性粒吞噬红细胞以及多分叶核巨核细胞改变的发生率前者高于后者（P＜0.05）；i(20q-)组与20q-伴随其它染色体异常组相比，少颗粒及空泡化中性粒细胞异常发生率两者相似，其它三种异常发生率前者高于后者（P＜0.05）。三组患者骨髓细胞中红系、粒系其它形态学异常的发生率无明显差异，但i(20q-)组易见到红系中的核碎裂、类巨幼变、空泡改变以及粒系中的颗粒减少等异常。在巨核系异常中，i(20q-)组与20q-伴其它异常组发生率之间无显著性差异，但二者分别与单纯20q-组相比，发现小巨核发生率前二者均较后者高，三系病态细胞比例(PMCC)比较发现，巨核系中，三组PMCC>10%的细胞比例i(20q-)组最高（P＜0.05），提示i(20q-)异常核型患者骨髓细胞病态造血更易涉及巨核系细胞。红系及粒系中20q-伴其它异常组患者的PMCC>10%的细胞比例最高，20q-伴其它异常组在二系中病态造血细胞比例最高，可能与该组患者核型比较复杂有关。生存分析显示，单纯20q-异常预后良好，20q-伴其它异常预后不良，i(20q-)预后介于两者之间。3、20q-异常血液病患者DNA芯片比较基因组（aCGH）杂交研究21例患者骨髓细胞全基因组aCGH检测结果示，17例均有20号染色体长臂中间缺失，8例为不连续的两部分缺失，9例为连续性大片段缺失。10例MDS中缺失片段重叠区域为20q11.23；4例AML中重叠区域为20q11.22-q13.2；2例ALL中重叠区域为20q11.21-q13.13；1例MPN缺失片段为20q11.21-q13.2。我们共筛选出三个共同缺失区域（common deleted region,CDR），CDR1位于20q11.23（14/17例），核苷酸起始点和终止点分别为34560497和37608229，片段大小3.05Mb；CDR2位于20q12(16/17例)，核苷酸起始点和终止点分别为37851501和39615698，片段大小约为1.76Mb；CDR3位于20q13.13(14/17例)，核苷酸起始点和终止点分别为48120412和48236791，片段大小约为116Kb。对20号染色体长臂保留区域进行分析，共筛选出两个共同保留区域（common retained region,CRR），CRR1位于20q11.21(29374726-30428250),大小1.1Mb，CRR2位于20q13.33(60484668-62963548),大小2.5Mb。通过UCSC GenomeBrowser及UniGene网站，发现CDR1上存在33个基因，CDR2上1个基因，CDR3上0个基因，CRR1上5个基因、CRR2上38个基因在骨髓或血细胞中有表达。4、六例20q-继发t(20;21)(q11;q11)易位的恶性血液病的临床和实验室特点6例20q-继发的t(20;21)异常占2001年以来20q-异常的2.3%，6例患者均经各种探针FISH证实存在20q-，其与21号染色体易位断裂点也均为20q11。患者以老年患者为主(中位年龄55岁)，多见于髓系疾病，尤其是MDS(5例MDS，1例ALL)，预后不良。结论20q-异常主要发生于髓系疾病，淋系疾病罕见，患者以中老年男性为主。对于常规核型检测怀疑存在20q-异常的患者，可以应用FISH技术加以验证。单纯20q-中位生存期较长，当伴随其他染色体异常同时存在时，即使是增加一条染色体异常，预后也较单纯20q-为差；20q-小克隆的中位生存时间长于大克隆，因此对20q-克隆群体大小变化的随访，可以提供一定的预后信息。i(20q-)患者骨髓细胞尽管无特异性形态学改变，但某些形态学改变的发生率明显高于其他核型异常，因此当发现以上某些特征性形态学改变时，应该首先考虑到i(20q-)的可能性较大。我们应用aCGH技术在20q-患者中确定了3个共同缺失区域及2个共同保留区域，目前仍无法确定20q-在不同疾病种类间是否涉及不同的基因从而导致截然不同的临床和实验室特征，需要进一步实验来揭示候选基因从而阐明致病机制，从而为寻找新的治疗靶点提供信息。der(20)del(20)(q11q12)t(20;21)(q11;q11)与i(20q-)一样，是20q-的另外一种较少见的再现性衍生异常。多见于中老年患者和髓系疾病，尤其是MDS，少数也可见于ALL。该类疾病预后不良，易位可能形成新的融合基因从而在该类疾病中起重要作用。更多还原

【Abstract】 Objectives:1.To analyse systematically the clinical and laboratorial characteristics of257hematological malignancy patients with20q-abnormality who received conventionalcytogenetics (CC) analysis in our laboratory from2001to2011and to reveal the uniqueclinical,cytogenetic and prognostic features of these patients in our area.2.To investigatewhether the i(20q-) harbors unique morphological features of the bone marrow cells andprognostic features.3.To analyse if there have significant differences in deletedregions,common deleted regions and amplified regions between different hematologicalmalignancies with20q-abnormality,so as to provide certain information to the analysis oftumor genes within these abnormal DNA copy number regions.4.To understand the clinicaland laboratorial characteristics of hematological malignancy patients with t(20;21)(q11;q11)abnormality which derived from20q-.Methods:1.257cases were studied by R-band karyotypic analysis using direct method and/orshort-term culture for chromosomes preparation.The20q11/12double color FISH probewas used to detect the chromosome abnormalities of the196cases which have thechromosomes reserved.All patients were classified according to the FAB proposals,ofwhich109cases were followed up to take the Kaplan-Meier survival analysis.2.The panelFISH probes,including20q12and20qter were used to detected the abnormality of i(20q-),a retrospective analysis about dysplasias of bone marrow cells was taken between thei(20q-),the isolate20q-,and the20q-accompanied by other chromosomal abnormalitieswho have the bone marrow slides reserved,survile analysis was performed between thethree groups.3.21cases whose cell samples were reserved were sended to ShangHai Biocorporation to take the whole genomic aCGH analysis,ues the normal bone marrow as the experimental control.4.The panel FISH probes,including20q11/12，SE20，SE21，20qter，21qter，WC20，and WC21were used to detected the abnormalities of t(20;12).Results:1.The clinical and cytogenetic features of257cases of hematologic malignancieswith20q-abnormalities257patients(163males/94females) with20q-abnormalty were registered in thisstudy,the ages were mainly in50-59years and60-69years.They suffered from:MPN(n=13)(5.1%),MDS(n=127)(49.4%),AML(n=45)(17.5%),CML(n=5)(2.0%),ALL(n=15)(5.8%),AL(n=6)(2.3%),Eosinophilicsyndrome(n=2)(0.8%),CLL(n=2)(0.8%),MH(n=1)(0.4%),macroglobulinemia(n=1)(0.4%),diagnostic unknown(n=40)(15.6%).CC analyses was performed on all257patients,of which,120patients(46.7%) wereisolate20q-,32patients(12.5%) were20q-accompanied by another one chromosomeabnormalities,72patients(28.0%) were complicated abnormalities with20q-,28patients(10.9%) were i(20q-),5patients(1.9%) were duplication of20q-. The male was domination,no significant differences between the groups were found for ages,median WBC counts,Hb level, PLT counts（P＞0.05）.In the group of20q-accompanied by other chromosomeabnormalities,the other23chromosomes were all involved,the abnormalities ofchromosome5were most frequent(41%),and then the chromosome7(40%) andchromosome9(39.7%) followed.In the FISH analysis of the196patients,there were almosthalf of the patients whose signal was1G1R,demonstrate that both20q11and20q12weredeleted,and there were half the patients whose positive cell percentages were extend50%,as the clone groups are large.As the survival analysis illustrated,the median survivalwas46months for the isolate20q-group,longer than the20q-accompanied by otherchromosome abnormalities group whose median survival was11months（P=0.00）.andlonger than the20q-accompanied with one chromosome abnormalities whose mediansurvival was14months （P=0.001）.No significant differences between the20q-accompanied with one chromosome abnormalities group and20q-complicated group.Themedian survival of20q-with complicated numeral and structural chromosomeabnormalities was11months,which longer than the complicated20q-with isolate structural abnormalities whose median survival was4months（P＜0.05）.The mediansurvival of small clones was longer than the large clones of20q-（P＜0.05）. No significantdifferences between2G1R signal group and1G1R signal group in median survival.2.The morphological and prognostic studies of idic(20q-)28patients(17males/11females) with i(20q-) abnormalty were registered in ourstudy,accounting for10.9%of the20q-patients from2001to2011.The incidences ofhypogranulated and vacuolized neutrophils, hypogranular and vacuolized eosinophils,neutrophil erythrophagocytosis, deeply lobulated and hyperlobulated megakaryocytes inbone marrow cells was higer in i(20q-) group than in isolate20q-group(P＜0.05). Nosignificant differences between i(20q-) group and20q-accompanied by other chromosomeabnormalities group in frequency of hypogranulated and vacuolized neutrophils,theincidences of other three type of morphological changes were higher in i(20q-) group（P＜0.05）. No significant differences between the three groups in frequency of the othererythroid or myeloid morphological changes, the incidences of nuclear fragmentation,megaloblastic change, vacuolized changes and hypogranulated changes in i(20q-) groupwere higher than in other two groups. In megakaryocyte,the incidence of smallmegakaryocyte in i(20q-) group and20q-accompanied by other chromosomeabnormalities group were both higher than the isolate20q-group. The incidence ofmorphological changes in megakaryocyte which PMCC＞10%was most frequent ini(20q-) group,suggest that the bone marrow cells of i(20q-) group were easily to involvedin megakaryocyte. The incidence of morphological changes in erythroid and myeloidwhich PMCC＞10%was most frequent in20q-accompanied by other chromosomeabnormalities group,it may resulted from the complexity of the kayrotype. The mediansurvival time of isolate20q-group is longer than the i(20q-) group,and the20q-accompanied by other chromosome abnormalities group has the shortest median survivaltime.3.The DNA aCGH study on hematoligic malignancies with20q-abnormalityThe whole genomic aCGH analysis of21cases showed that,17cases had interstitial deletion of the long arm of chromosome20,of which8cases were two consecutive partialdeletions,9cases were continuity of large deletions.The overlap region of deleted regionsin the10cases of MDS was20q11.23, the overlap region of deleted regions in4cases ofAML was20q11.22-q13.2, the overlap region of deleted regions in2cases of ALL was20q11.21-q13.13,the deleted region in MPN was20q11.21-q13.2.We defined three CDRsand two CRRs, CDR1(14/17patients) spanned3.05Mb and was located in20q11.23(34,560,497-37,608,229). CDR2(16/17patients) spanned1.76Mb and waslocated in20q12(37,851,501-39,615,698). CDR3(14/17patients) spanned116kb and waslocated in20q13.13(48,120,412-48,236,791). CRR1(14/17patients)spanned1.1Mb, locatedin20q11.11(29,374,726-30,428，250).CRR2(14/17patients) spanned2.5Mb and waslocated in20q13.33(60,484,668-62,963,548). we used the UCSC Genome Browser andBioGPS based on their function and expression pattern (expression in bone marrow orblood cells) to pinpoint putative pathogenic genes of interest. CDR1,CDR2and CDR3spanned33、1and0genes known to be expressed in hematopoietic tissues, while CRR1and CRR2encompassed5and38genes, respectively.4.The clinical and laboratorial features of hematologic malignancies with t(20;21)The6cases with t(20;21) abnormalty accounting for2.3%of the20q-patients from2001.A panel FISH probes confirmed the existence of the20q-.The translocation breakingpoints of chromosome20which was translocated with chromosome21was20q11.Theelderly patient was domination(median age was55years old), and20q-is mainlyassociated with myeloid malignancies,especially with MDS(5cases of MDS,1case ofALL), the outcome is usually poor.Conclusions:Deletion of the long arm of chromosome20is a common abnormality associated withmyeloid malignancies,it is rarely seen in lymphoid malignancies,the elderly male weredomination. We can perform FISH test for those patients suspected of20q-by CC.Themedian survival of isolate20q-is relatively longer, but the outcome is inferior even ifone additional chromosome abnormality is present.The median survival in20q-smallclones is longer than in the large clones.so it can provide some prognostic information by following up the clone population changes.Although there’s no specific bone marrow morphological changes in i(20q-), theincidences of some morphological changes were significantly higher than in otherchromosome abnormalities groups,when we found those certain characteristicmorphological changes,we should take i(20q-) into account first.We defined3CDRs and2CRRs in20q-patients by aCGH, but it is still unknown thatwhether20q-cause the loss of several different tumor suppressor genes which result in thedistinct clinical and laboratorial features. So we need further study to reveal the pathogenicmechanism of the candidate genes.so as to provide some information to therapeutic targets.As i(20q-),der(20)del(20)(q11q12)t(20;21)(q11;q11) is a rare but recurringabnormality which derived from20q-, the elderly male patient was domination,and ismainly associated with myeloid malignancies,especially in MDS,rarely occurred inALL.The outcome of this abnormalty is poor. Translocation may play an important role insuch disorders by causing a new fusion gene.更多还原