用定量多色荧光原位杂交检测t（8；21）急性髓系白血病的异质性及亚克隆间的进化关系  

Detection of heterogeneity and evolution of subclones in t （ 8;21 ） AML by QM-FISH

作　　者：王英婵 胡林萍[1,2] 林冬[1,2] 李承文[1,2] 袁田[1,2] 贾玉娇[1,2] 田征[1,2] 唐克晶[1,2] 王敏[1,2] 王建祥[1,2] 

机构地区：[1]中国医学科学院、北京协和医学院血液学研究所、血液病医院,天津300020 [2]实验血液学国家重点实验室,天津300020

出　　处：《中华血液学杂志》2013年第10期844-850,共7页Chinese Journal of Hematology

基　　金：天津国际合作项目（09ZCZDSF03800）;国家科技重大专项（2011ZX09302-007-04）

摘　　要：目的用定量多色荧光原位杂交（QM．FISH）的方法检测t（8；21）急性髓系白血病（AML）中是否存在异质性亚克隆，及不同亚克隆之间在遗传学上的承继关系。方法在UCSC Genome Bioinformatics数据库中定位检索含目的基因AMLl、ETO、WTl、c-kit和p27的细菌人工染色体（BAC）克隆，用缺口平移的方法将荧光素染料标记的dUTP或dCTP连接到目的基因上制成五色FISH探针。白血病细胞低渗、同定后湿法滴片。将制备好的FISH探针杂交到患者标本上，经荧光显微镜采集荧光信号从而获得清晰稳定的多色图像。计数AMLl．ETO阳性细胞中AMLl-ETO融合基因及基因AMLl、ETO、WTl、c-kit和p27的拷贝数。将各个基因拷贝数相同的细胞定义为同一亚克隆，统计同一患者标本中亚克隆的种类及比例，进而推测不同亚克隆之间的进化关系；比较配对的初诊、复发患者中优势亚克隆的演变，推测与白血病复发、耐药相关的基因异常。结果共检测36例初诊的t（8；21）AML患者骨髓标本，1例与初诊患者配对的复发标本。在36例初诊患者中，4例（11．1％）具有AML1-ETO融合信号增加，3例（8．3％）有AMLl信号增加，10例（27．8％）有AMLl信号缺失，并且这种信号缺失具有性别差异，主要出现在男性患者中，4例（11．1％）有ETO信号增加，20例（55．6％）有wTl信号增加，15例（41．7％）有p27信号增加，14例（38．9％）有c-kit信号增加；28例（77．8％）可检测到异质性亚克隆，不同亚克隆之间呈线性或树状进化关系；1例复发病例标本与配对的初诊病例标本相比较，表现出明显的克隆演变，包括优势克隆比例改变及亚克隆的消失与新生。结论t（8；21）AML的遗传学异常具有广泛异质性，亚克隆之间不仅具有承继关系，而且处于动态进化演变状态。Objective To explore the heterogeneous subclones in acute myeloid leukemia （AML） with t （ 8;21 ） by quantitative multicolor- fluorescence in situ hybridization （QM- FISH）, and to figure out whether there is putative ancestral relationship among different subclones. Methods Bacterial artificial chromosomes （BAC） clones that contain the targeted genes including AML1, ETO, WT1, p27 and c-kit were searched in the data base UCSC Genome Bioinformatics. Multicolor FISH probes were prepared by linking fluorescein labeled dUTP or dCTP to targeted genes by nick translation. Bone marrow mononuclear cells from t （8;21） AML patients are dropped on to the wet surface of glass slides after hypotonic treatment and fixation. After hybridization, the fluorescence signals were captured by Zeiss fluorescence microscope. The copy number of AML1, ETO, WT1, p27, c-kit and the AML1-ETO fusion gene in AML1-ETO positive cells was counted. The cells with same signals were defined as a subclone. Various subclones were recorded and their proportions were calculated, and their evolutionary relationship was deduced. The subclones in matched primary and relapsed samples were compared, the evolution of dominant clones were figured out and the genomic abnormality that is associated with relapse and drug resistance were speculated. Results In this study, 36 primary AML with t（8;21 ） cases and 1 relapsed casepaired with the primary case were detected. In these 36 primary cases, 4 cases （11.1%） acquired additional AM LI-ETO fusion signal, 3（8.3%） had additional AML1 signal, 4 （ 11.1% ） had additional ETO signal, 20 （55.6%） had additional WTI signal, 15 （41.7%） had additional p27 signal and 14 （38.9%） had additional c-kit signal. In addition, 10 （27.8%） displayed AMLI signal deletion, and such an aberration represents statistic significance in male patients. It seems that male patients usually accompany AMLI signal deletion. Of 36 cases, 28 （77.8 %） harbored at least 2 subclones （rauged from 2

关 键 词：白血病 髓样 急性 AML1-ETO融合基因 原位杂交 荧光 克隆进化 

分 类 号：R733.71[医药卫生—肿瘤]