双色双融合与额外信号探针在BCR/ABL融合基因检测中的比较及其意义

目的 探讨DCCF(dual color dual fusion)探针与ES(extra signal)探针在BCR/ABL融合基因检测中信号表现的特点,并明确其信号特征与染色体核型的相互关系.方法 对初治65例慢性粒细胞白血病(chronic myelocytic leukemia,CML)及50例急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)患者骨髓标本进行BCR/ABL的DCDF探针的荧光原位杂交(fluorescence in situ hybridization,FISH)检测,FISH阳性标本则使用ES探针再次进行荧光原位杂交进行BCR断裂点的检测,同时对其中47例CML和40例ALL进行了核型分析.结果 FISH结果示65例CML均为BCR/ABL阳性,DCDFFISH中有17例为非典型信号表现,ES-FISH有12例为非典型信号表现;50例ALL中7例BCR/ABL阳性,ES探针显示5例BCR断裂点位于m-bcr,2例位于M-bcr.核型分析CML检出Ph阳性98%(43/44),ALL检出Ph阳性22%(7/32).结论 DCDF-FISH、ES-FISH以及核型分析各有其特性.根据每种方法的特性,对实验结果进行综合分析可对遗传学特征作出更准确判断.     

双色双融合与额外信号探针在BCR/ABL融合基因检测中的比较及其意义

目的 探讨DCCF(dual color dual fusion)探针与ES(extra signal)探针在BCR/ABL融合基因检测中信号表现的特点,并明确其信号特征与染色体核型的相互关系.方法 对初治65例慢性粒细胞白血病(chronic myelocytic leukemia,CML)及50例急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)患者骨髓标本进行BCR/ABL的DCDF探针的荧光原位杂交(fluorescence in situ hybridization,FISH)检测,FISH阳性标本则使用ES探针再次进行荧光原位杂交进行BCR断裂点的检测,同时对其中47例CML和40例ALL进行了核型分析.结果 FISH结果示65例CML均为BCR/ABL阳性,DCDFFISH中有17例为非典型信号表现,ES-FISH有12例为非典型信号表现;50例ALL中7例BCR/ABL阳性,ES探针显示5例BCR断裂点位于m-bcr,2例位于M-bcr.核型分析CML检出Ph阳性98%(43/44),ALL检出Ph阳性22%(7/32).结论 DCDF-FISH、ES-FISH以及核型分析各有其特性.根据每种方法的特性,对实验结果进行综合分析可对遗传学特征作出更准确判断.     

应用荧光原位杂交技术检测慢性粒细胞白血病的融合基因

目的 应用荧光原位杂交技术(FISH)直接检测慢性粒细胞白血病(CML)的融合基因(BCR/ABL)以辅助临床诊断和治疗白血病.方法 应用FISH技术检测25例CML自1991年-2008年制备染色体剩余细胞悬液的BCR/ABL,6例是2008年的标本,7例是1991年-1995年的标本,余10例标本是2001年-20...     

双色双融合与额外信号探针在BCR/ABL融合基因检测中的比较及其意义

目的 探讨DCCF(dual color dual fusion)探针与ES(extra signal)探针在BCR/ABL融合基因检测中信号表现的特点,并明确其信号特征与染色体核型的相互关系.方法 对初治65例慢性粒细胞白血病(chronic myelocytic leukemia,CML)及50例急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)患者骨髓标本进行BCR/ABL的DCDF探针的荧光原位杂交(fluorescence in situ hybridization,FISH)检测,FISH阳性标本则使用ES探针再次进行荧光原位杂交进行BCR断裂点的检测,同时对其中47例CML和40例ALL进行了核型分析.结果 FISH结果示65例CML均为BCR/ABL阳性,DCDFFISH中有17例为非典型信号表现,ES-FISH有12例为非典型信号表现;50例ALL中7例BCR/ABL阳性,ES探针显示5例BCR断裂点位于m-bcr,2例位于M-bcr.核型分析CML检出Ph阳性98%(43/44),ALL检出Ph阳性22%(7/32).结论 DCDF-FISH、ES-FISH以及核型分析各有其特性.根据每种方法的特性,对实验结果进行综合分析可对遗传学特征作出更准确判断.

Abstract：

Objective To investigate the signal patterns of dual color dual fusion (DCDF) probe and extra signal (ES) probe in the detection of BCR/ABL fusion gene, and illustrate the relation between the fluorescence in situ hybridization (FISH) pattern and the karyotype. Methods Sixty-five cases of chronic myelocytic leukemia(CML) and 50 cases of acute lymphoblastic leukemia (ALL) were detected by FISH with DCDF probe, the BCR/ABL positive samples were detected by FISH with ES probe. Among these cases, 47 cases of CML and 40 cases of ALL perform conventional cytogenetics simultaneously. Results All 65 cases of CML were all BCR/ABL positive by FISH. 17 cases showed the atypical pattern by DCDFFISH, and 12 cases showed the atypical pattern by ES-FISH. There were 7 cases of BCR/ABL positive in 50 cases of ALL by FISH. By ES-FISH, there were 5 cases in which the break-point of BCR gene was located in m-bcr, 2 cases in which the break-point of BCR gene was located in M-bcr. Conventional cytogenetics demonstrated that 43/44(98 %) cases of CML and 7/32(22 %) cases of ALL were Ph positive.Conclusion The features of DCDF-FISH, ES-FISH and conventional eytogenetic are different from each other. According to the features of these method, it can increase the precision of the adjustment of genetic feature to analyze these results comprehensively.     

荧光原位杂交检测慢性粒细胞白血病

目的 探讨对慢性粒细胞白血病进行荧光原位杂交(fluorescence in situ hybridization,FISH)检测的意义.方法 对158例慢性粒细胞白血病标本采用24 h短期培养法制备染色体,然后应用双色双融合BCR/ABL探针进行FISH检测,部分标本同时采用R显带技术进行染色体核型分析.结果 158例中共检出Ph阳性标本98例,其中69例(70.4%)为典型双色双融合BCR/ABL探针信号模式(1R1G2F),其余29例(29.6%)为3类12种非典型模式.各种非典型信号模式中出现频率较高的依次为:1R1G1F7例(7.1%)、2R1G1F 5例(5.1%)、1R1G2F&1R1G3F 4例(4.1%)、2R2G1F 3例(3.1%).对18例有核型资料的非典型信号的病例分析显示:其中3例特殊信号系由变异Ph易位引起;2例中出现的3个融合信号来源于附加的Ph染色体;4例核型与FISH结果不吻合,提示染色体分析存在错漏之处;3例染色体为典型Ph易位,而FISH结果为单个融合信号,系由der(9)号的部分缺失所致;3例核型中未发现Ph染色体.但FISH显示40%～64%的细胞中存在一个融合信号,从而明确慢性粒细胞白血病诊断;3例是移植或经格列卫治疗后的患者,染色体均为正常核型,而FISH检测到极小比例的阳性细胞.结论 FISH在慢性粒细胞白血病诊断、判断变异易位、隐匿Ph易位、衍生9号缺失、干扰素及格列卫的疗效观察以及移植后监测等诸多方面均具有重要价值.     

荧光原位杂交在儿童急性淋巴细胞白血病诊疗中的应用分析

目的研究荧光原位杂交技术(fluorescence ?in? situ ?hybridiza-tion,FISH)在儿童急性淋巴细胞白血病(acute ?lymphoblastic? leu-kemia,ALL)诊疗中的应用分析。方法应用荧光原位杂交技术对136例急性淋巴细胞白血病患儿用标记的MYC、P16、E2A、CHIC2/D10Z1/?D17Z1、TEL/AML1、MLL、BCR/ABL1、IGHDNA探针进行检测。结果荧光原位杂交技术在136?例患儿中总共检出了81例细胞遗传学改变,总体阳性率为59.56%,包括八种细胞遗传学异常。其中TEL/AML1融合基因和CHIC2/D1021/D1721多倍体检测中的阳性率较高,共43例,占53.10%,而BCR/ABL融合基因的阳性率最低,共3例,占4.70%。结论 FISH可以检出ALL的常见遗传学异常;TEL/AML1融合基因和CHIC2/D1021/D1721多倍体检测是儿童ALL最常见的分子遗传学异常,荧光原位杂交技术在儿童急性淋巴细胞白血病诊疗中显著提高检出率,在协助临床制定儿童ALL治疗方案及判断预后中具有重要价值。     

两种BCR/ABL探针在Ph阳性白血病荧光原位杂交检测中的不同信号模式及其意义

目的 比较BCR/ABL双色额外信号探针(dual color extra-signal BCR/ABL probe,ESFISH探针)及BCR/ABL双色双融合探针(dual color dual fusion BCR/ABL probe,D-FISH探针)在Ph阳性白血病荧光原位杂交(fluorescence in situ hybridization,FISH)检测中信号模式的差异,探讨它们的诊断价值.方法 分别采用D-FISH和ES-FISH探针对74例伴有单纯t(9;22)(q34;q11)及37例伴有变异Ph易位或复杂核型异常的Ph阳性白血病患者骨髓细胞进行间期FISH检测.结果 所有单纯t(9;22)(q34;q11)易位的白血病患者应用两种探针均检测到BCR/ABL阳性信号,ES-FISH探针显示2个橙色信号、1个绿色信号和1个黄色信号模式,而D-FISH探针显示1个橙色信号、1个绿色信号和2个黄色信号模式.ES-FISH探针在9例(12.2%)Ph阳性白血病患者中识别次要BCR断裂位点(1个橙色信号、1个绿色信号和2个黄色信号),而D-FISH探针不能识别主要BCR和次要BCR断裂位点;D-FISH探针在8例(10.8%)Ph阳性白血病中区分ABL基因单独缺失(1个橙色信号、2个绿色信号、1个黄色信号)和ABL、BCR基因共同缺失(1个橙色信号、1个绿色信号和1个黄色信号),ES-FISH则不能区分之.检测变异Ph易位和含Ph易位的复杂核型异常时,两种探针的信号模式分别有4种和6种之多,且以不典型者居多,对于它们的精确解释必须依赖常规染色体分析和中期FISH结果 .结论 ES-FISH及D-FISH探针由于BCR探针大小及覆盖区域不同,在Ph阳性白血病的FISH检测中显示不同信号模式,可分别作为Ph+急性淋巴细胞白血病和慢性髓系白血病患者FISH检测的首选.若采用伊马替尼治疗,主要BCR断裂点和次要BCR断裂点、伴或不伴有衍生9号染色体部分序列缺失均不影响预后,但鉴于ES-FISH探针性价比优于D-FISH探针,推荐其作为Ph阳性白血病FISH检测的首选.     

两种BCR/ABL探针在Ph阳性白血病荧光原位杂交检测中的不同信号模式及其意义

目的 比较BCR/ABL双色额外信号探针(dual color extra-signal BCR/ABL probe,ESFISH探针)及BCR/ABL双色双融合探针(dual color dual fusion BCR/ABL probe,D-FISH探针)在Ph阳性白血病荧光原位杂交(fluorescence in situ hybridization,FISH)检测中信号模式的差异,探讨它们的诊断价值.方法 分别采用D-FISH和ES-FISH探针对74例伴有单纯t(9;22)(q34;q11)及37例伴有变异Ph易位或复杂核型异常的Ph阳性白血病患者骨髓细胞进行间期FISH检测.结果 所有单纯t(9;22)(q34;q11)易位的白血病患者应用两种探针均检测到BCR/ABL阳性信号,ES-FISH探针显示2个橙色信号、1个绿色信号和1个黄色信号模式,而D-FISH探针显示1个橙色信号、1个绿色信号和2个黄色信号模式.ES-FISH探针在9例(12.2%)Ph阳性白血病患者中识别次要BCR断裂位点(1个橙色信号、1个绿色信号和2个黄色信号),而D-FISH探针不能识别主要BCR和次要BCR断裂位点;D-FISH探针在8例(10.8%)Ph阳性白血病中区分ABL基因单独缺失(1个橙色信号、2个绿色信号、1个黄色信号)和ABL、BCR基因共同缺失(1个橙色信号、1个绿色信号和1个黄色信号),ES-FISH则不能区分之.检测变异Ph易位和含Ph易位的复杂核型异常时,两种探针的信号模式分别有4种和6种之多,且以不典型者居多,对于它们的精确解释必须依赖常规染色体分析和中期FISH结果 .结论 ES-FISH及D-FISH探针由于BCR探针大小及覆盖区域不同,在Ph阳性白血病的FISH检测中显示不同信号模式,可分别作为Ph+急性淋巴细胞白血病和慢性髓系白血病患者FISH检测的首选.若采用伊马替尼治疗,主要BCR断裂点和次要BCR断裂点、伴或不伴有衍生9号染色体部分序列缺失均不影响预后,但鉴于ES-FISH探针性价比优于D-FISH探针,推荐其作为Ph阳性白血病FISH检测的首选.     

两种BCR/ABL探针在Ph阳性白血病荧光原位杂交检测中的不同信号模式及其意义

目的 比较BCR/ABL双色额外信号探针(dual color extra-signal BCR/ABL probe,ESFISH探针)及BCR/ABL双色双融合探针(dual color dual fusion BCR/ABL probe,D-FISH探针)在Ph阳性白血病荧光原位杂交(fluorescence in situ hybridization,FISH)检测中信号模式的差异,探讨它们的诊断价值.方法 分别采用D-FISH和ES-FISH探针对74例伴有单纯t(9;22)(q34;q11)及37例伴有变异Ph易位或复杂核型异常的Ph阳性白血病患者骨髓细胞进行间期FISH检测.结果 所有单纯t(9;22)(q34;q11)易位的白血病患者应用两种探针均检测到BCR/ABL阳性信号,ES-FISH探针显示2个橙色信号、1个绿色信号和1个黄色信号模式,而D-FISH探针显示1个橙色信号、1个绿色信号和2个黄色信号模式.ES-FISH探针在9例(12.2%)Ph阳性白血病患者中识别次要BCR断裂位点(1个橙色信号、1个绿色信号和2个黄色信号),而D-FISH探针不能识别主要BCR和次要BCR断裂位点;D-FISH探针在8例(10.8%)Ph阳性白血病中区分ABL基因单独缺失(1个橙色信号、2个绿色信号、1个黄色信号)和ABL、BCR基因共同缺失(1个橙色信号、1个绿色信号和1个黄色信号),ES-FISH则不能区分之.检测变异Ph易位和含Ph易位的复杂核型异常时,两种探针的信号模式分别有4种和6种之多,且以不典型者居多,对于它们的精确解释必须依赖常规染色体分析和中期FISH结果 .结论 ES-FISH及D-FISH探针由于BCR探针大小及覆盖区域不同,在Ph阳性白血病的FISH检测中显示不同信号模式,可分别作为Ph+急性淋巴细胞白血病和慢性髓系白血病患者FISH检测的首选.若采用伊马替尼治疗,主要BCR断裂点和次要BCR断裂点、伴或不伴有衍生9号染色体部分序列缺失均不影响预后,但鉴于ES-FISH探针性价比优于D-FISH探针,推荐其作为Ph阳性白血病FISH检测的首选.     

荧光原位杂交技术在诊断变异Ph易位及Ph(一)慢性髓细胞白血病中的应用

目的 探讨荧光原位杂交(fluorescence in situ hybridization,FISH)技术在诊断变异Ph易位及Ph(-)慢性髓细胞白血病(chronic myelocytic leukemia,CML)中的应用价值.方法 应用常规R显带方法,对9例伴有变异Ph易位和2例Ph(-)CML患者采用双色双融合bcr/abl探针进行FISH检测.结果 9例变异Ph易位CML患者异常核型除涉及9和22号染色体外,还涉及1、3、5、12、13、15、17、21号染色体,且部分类型为重现性异常,FISH结果均为阳性,信号特征为2R2G1Y;2例Ph(-)CML患者核型正常,FISH结果阳性,信号特征分别为1R1G2Y和1R1G1Y.结论 FISH技术对伴有变异Ph易位及Ph(-)CML患者的诊断更具有优势,可根据阳性细胞信号特征分析其异常核型,判断标记基因异常改变情况,是常规染色体显带分析的有益补充.     

A simple strategy for breakpoint fragment determination in chronic myeloid leukemia

Molecular characterization is considered a part of the routine work-up of chronic myeloid leukemia (CML) cases. Southern blot analysis using the universal BCR (UBCR) probe on BglII-digested DNA samples is the most commonly used technique, while employing the human 3' bcr probe (PR-1) is usually considered a complementary tool. In this study, we tried to develop a simple and economic strategy for molecular characterization of CML using the 3' probe as it has been shown to be the one capable of locating the breakpoint site. Seventy-eight cases of CML were studied. Molecular analysis was performed using the Southern blot technique. DNA was digested with Bam HI, BglII, EcoRI, and XbaI. Hybridization was performed using the human 3' bcr (PR-1) probe. BamHI and BglII could differentiate fragment 1 (F1) showing rearrangement (R) with Bam HI and germline configuration (G) with BglII; F2/3 showing R with both, and F4 showing R with BamHI and G with BglII. F2/3 cases were further divided by HindIII enzyme into F2 showing (G) and F3 showing (R). Fragment 0 showed G with both, but R with EcoRI and/or XbaI, while 3' deletion gave G with all four enzymes. Our results showed a relative incidence of 6.4% for F0, 20.5% for F1, 32.1% for F2, 19.2% for F3, 15.4% for F4, and 6.4% for 3' deletion. Sixty cases were evaluated clinically and hematologically and were followed up for disease evolution and survival. They included 32 cases in early chronic phase, 24 in late chronic phase, two in acceleration, and two in blastic crisis. No significant correlation was encountered between the breakpoint site and any of the clinical and hematological data except those patients with 3' deletion who showed a very short survival. The study emphasizes Southern blotting as the method of choice for molecular characterization of CML and offers a simple and economic strategy for diagnosis and determination of breakpoint fragment.     

BCR-ABL探针在慢性粒细胞白血病中的应用

目的用常规细胞遗传学（conventional cytogenetics,CC）和荧光原位杂交（fluorescence chromosomal in situ hybridization,FISH）技术检测Ph染色体。方法常规细胞遗传学分析（CC）,荧光原位杂交（FISH）技术。结果7例患者4例Ph染色体阴性,其中2例分别伴有t（18;22）和t（17;22）异常,其余2例为异基因造血干细胞移植后正常核型。一例培养后无中期分裂相。2例Ph染色体阳性,FISH结果bcr/abl（＋）细胞检出率分别为63.87%,84.51%,7.56%,4.0%,74.45%,67%,47%。结论常规细胞遗传学与荧光原位杂交技术相结合对CML患者诊断治疗过程中肿瘤负荷动态检测有显著意义。     

A two-color BCR–ABL probe that greatly reduces the false positive and false negative rates for fluorescence in situ hybridization in chronic myeloid leukemia

The t(9;22) translocation resulting in the fusion of BCRand ABLgenes is pathognomonic in chronic myeloid leukemia (CML) and may be investigated at the molecular level using fluorescence in situ hybridization (FISH). Two-color BCR–ABL probes visualizing one fusion signal (1F FISH) have high false positive rates (FPR) and false negative rates (FNR). The FPR is a result of the random spatial association of probe signals within normal interphase cells so that some cells appear to contain the BCR–ABLfusion gene. The FNR of 1F FISH probes depends on the distance between the BCR and ABL probes hybridized to the BCR–ABL fusion gene (≤368 kb); the “gap” between the signals causing the cell to be interpreted as normal. To overcome these difficulties, a two-color probe was used, employing four yeast artificial chromosome (YAC) sequences that span the breakpoint regions of the BCR and ABL genes and that visualize the two fusion signals BCR–ABL and ABL–BCRin CML cells (2F FISH). The FNR for the 2F FISH probes was assessed on clonal Ph+ granulocyte-macrophage-colony-forming cell (CFU-GM) derived colonies and was reduced to 0.4% (2/450), compared with an FNR of 13.5% (111/823) with 1F FISH. The FPR in normal mononuclear cells for the 2F FISH was 0.19 ± 0.12% (3/1,700), whereas the FPR using 1F FISH was 4.5 ± 2.3% (63/1,294). The 2F FISH can thus be used to evaluate very small frequencies of BCR–ABL-positive and -negative interphase cells and may be of use in the clinical monitoring of CML. Genes Chromosomes Cancer 23:109–115, 1998. © 1998 Wiley-Liss, Inc.     

A FISH study of variant Philadelphia rearrangements

A total of 39 variant Philadelphia (Ph) translocations were studied by fluorescence in situ hybridization (FISH) using MBCR/ABL, mBCR/ABL, or DBCR/ABL probes. Seven cases did not have a BCR/ABL fusion signal. Of a total of 32 fusion-positive cases, 5 were simple variants involving chromosome 22 and another chromosome apart from chromosome 9; 23 were complex variants involving chromosomes 22, 9, and a third chromosome (18 cases), or 22, 9, and two other chromosomes (4 cases). Masked Ph rearrangements were detected in 4 cases. One case was a Ph chromosome mimic. Fluorescence in situ hybridization has become a widely used method for studying Ph rearrangements. The latest probe that is being used is the DBCR/ABL (double reciprocal BCR/ABL signals). The expected pattern for this probe is one green ABL signal (1G) on the normal 9, one red BCR signal (1R) on the normal 22, and two fusion signals, BCR/ABL and ABL/BCR (2F), on a derivative 22 and a derivative 9, respectively. Deviant patterns from 1G1R2F, and sometimes 1G1R2F, were indicative of a variant, as long as there was a fusion signal. However, in interphase analysis, it is not possible to visualize a variant rearrangement, and when a deviant pattern involving at least one fusion signal is observed, the following possibilities should be contemplated. The different patterns observed in fifteen Ph variants are described. The patterns observed in variants studied with the DBCR/ABL probe were 2G2R1F (40%), 1G1R2F (20%), 1G1R1F (20%), 1G2R1F (13.3%), and 2G1R1F (6.66%). A single mechanism is involved in the formation of each of these patterns. A 2G2R1F, FISH pattern in 6 cases appears to involve a single concerted event of simultaneous breaks on the participating chromosomes followed by mismatched joining. The three cases with 1G1R2F most probably arose by two sequential rearrangements. The 1G1R1F pattern suggests that either the BCR and ABL breakpoints are different, or there are deletions at the breakpoints, because residual signals are not observed. Two independent events appear to be involved in 1G2R1F with a reverse cryptic 9,22 rearrangement as the first event. In one case of 2G1R1F, the plausible explanation is an insertion of ABL next to BCR and either a simultaneous or a sequential translocation with another chromosome.     

荧光原位杂交技术检测慢性粒细胞白血病微小残留病

目的 研究应用荧光原位杂交技术(FISH)检测慢性粒细胞白血病微小残留病，及用FISH技术对缓解期慢性粒细胞白血病(CML)患者外周血进行检测，评价其体内微小残留病的意义。方法 应用CG和I-FI舛对30例CML(13例给予化疗、17例移植后)患者初发和／或缓解期的骨髓及外周血标本进行分析，分别检测Ph染色体和BCR/ABL融合基因的存在。结果 对13例临床给予化疗的患者初发期的外周血和骨髓进行CG分析，Ph检出率分别为15％(2／13)、100％(13／13)。同时进行I-FISH分析，均可检出BCR/ABL融合基因。对初发期骨髓的CG、I-FISH分析结果进行统计学分析，两组无显著差异；对外周血的CG、I-FISH分析结果进行统计学分析，两组有显著差异。对其缓解期骨髓CG、I-FISH分析结果进行统计学分析，两组有显著差异；对缓解期外周血CG、I-FISH结果进行统计学分析，两组有显著差异；对缓解期外周血I-FISH和骨髓I-FISH结果进行统计学分析，两组呈显著相关。对17例移植后患者CG、I-FISH结果进行统计学分析，两组有显著差异。结论 FISH技术检测慢性粒细胞白血病微小残留病敏感性大大高于常规细胞遗传学分析；采集缓解期外周血进行荧光原位杂交分析，可作为一种方便易行的手段评价微小残留病。