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.     

A novel tricolor, dual-fusion fluorescence in situ hybridization method to detect BCR/ABL fusion in cells with t(9;22)(q34;q11.2) associated with deletion of DNA on the derivative chromosome 9 in chronic myelocytic leukemia

Dual-color, dual-fusion fluorescence in situ hybridization (D-FISH) can accurately detect and quantify cells with BCR/ABL fusion in <1% of 500 nuclei in 80% of patients with chronic myelocytic leukemia (CML) and t(9;22)(q34;q11.2). The remaining patients have one of three forms of atypical D-FISH patterns; these patterns have different sensitivities to detect disease. Neoplastic cells with one ABL, one BCR, and one BCR/ABL fusion are particularly problematic, because normal cells with coincidental overlap have the same pattern. For these patients, the normal cutoff for D-FISH is >23%. We tested a new method that incorporates an aqua-labeled probe for the argininosuccinate synthetase (ASS) gene into the conventional BCR/ABL D-FISH probe set. This tricolor D-FISH (TD-FISH) method takes advantage of the aqua-labeled ASS probe to distinguish between neoplastic and normal cells. We used TD-FISH to study 20 normal specimens and 35 specimens from 20 patients with known loss of both BCR and ABL from the derivative chromosome 9. The results show that TD-FISH effectively discriminates between cells with overlapping BCR and ABL signals from cells with true BCR/ABL fusion and improves the ability to quantify minimal residual disease from >23% to >1% of 500 interphase nuclei.     

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.     

Interpretation of submicroscopic deletions of the BCR or ABL gene should not depend on extra signal-FISH: problems in interpretation of submicroscopic deletion of the BCR or ABL gene with extra signal-FISH

Several groups have demonstrated that a submicroscopic gene deletion in Ph+ chronic myelogenous leukemia (CML) is associated with a poor prognosis and reduced response to treatment. To assess the variation between detection methods in the interpretation of a submicroscopic gene deletion, we performed an extra signal (ES)-FISH BCR/ABL and double-FISH (D-FISH) BCR/ABL on frozen bone marrow cells from 79 patients with CML (63 in the chronic phase, 6 in the accelerated phase, and 10 in blast crisis) and 30 patients with a BCR/ABL-negative myeloproliferative disorder as determined by RT-PCR. The normal cutoff values were 0.22% for ES-FISH and 0.25% for D-FISH. The cutoff values for false-positive signals from a juxtaposition of the BCR and ABL gene were 11% in ES-FISH and 13% in D-FISH. Of the 14 patients who showed an ABL gene deletion by ES-FISH, 5 had an ABL deletion only, 5 had both a BCR and an ABL deletion, but 4 proved to have a classic BCR/ABL rearrangement without a submicroscopic deletion, as determined by D-FISH. Discrepant results between ES- and D-FISH were observed in 12 of the 79 patients (15.8%), and the main causes of a discrepancy were a false-positive ABL deletion (4 of 12, 33%), a variant Philadelphia chromosome (3 of 12, 25%), an inversion of derivative chromosome 9 at the very breakpoint of the ABL gene (9q32) (1 of 12, 8.3%), a cryptic variant Ph chromosome (1 of 12, 8.3%), and a marker chromosome (1 of 12, 8.3%). Although there was no significant difference in the sensitivity for the detection of the fusion signal between ES- and D-FISH, ES-FISH showed a high percentage of cells with false-positive fusion signals (1 orange, 1 green, 1 yellow), which makes it difficult to interpret the submicroscopic ABL deletion. In conclusion, an interpretation of the submicroscopic deletions of the BCR or ABL gene should not depend on ES-FISH.     

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.

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.     

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.     

Interphase fluorescence in situ hybridization studies for the detection of 9q34 deletions in chronic myelogenous leukemia: a practical approach to clinical diagnosis

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome (Ph) in more than 90% of cases. Recent studies using fluorescence in situ hybridization (FISH) have shown that in a subset of patients with CML, deletions of 9q34 involving the argininosuccinate synthetase region occur at the time of the Philadelphia translocation and are associated with a poor prognosis. We performed interphase FISH studies in 152 cases of CML using a dual-color, dual-fusion probe system with a third probe directed at 9q34. Cytogenetic studies showed a simple (typical) Ph in 124/152 (82%), a cryptic Ph in 11/152 (7%), and a variant Ph chromosome with a complex translocation in 17/152 (11%) of cases. Interphase FISH studies showed single BCR/ABL fusion patterns in 48/152 (32%) of cases. Deletions of 9q34 were observed in 14% of all the cases and were present in 46% of cases with single BCR/ABL fusion pattern. All the 9q34 deletions occurred in cases with single BCR/ABL fusion signal. However, a single-fusion pattern is not specific for 9q34 deletions, and cases should be routinely screened for the presence of this prognostically significant abnormality by using a third probe directed specifically at 9q34.     

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...     

Combined analysis of morphology and fluorescence in situ hybridization in follow-up of minimal residual disease in a child with Philadelphia-positive acute lymphoblastic leukemia

The past decade has brought new technologies to the study of minimal residual disease (MRD) in leukemia. Each of them has limitations and is far from being accurate. Recently, a new multiparametric cell scanning system (Duet) was introduced to the field of MRD detection. This system has the advantage of automatically scanning large numbers of cells and performing combined analysis of morphology and fluorescence in situ hybridization (FISH) on the same cell. We used this system to characterize the lineage and degree of maturation of the cells carrying the minor m-BCR/ABL fusion, in a follow-up of an 8-year-old boy with Philadelphia-positive (Ph(+)) acute lymphoblastic leukemia (ALL). The boy was treated using a high-risk protocol and was closely monitored with FISH analysis for cells carrying the m-BCR/ABL fusion. Consecutive analysis along 2.5 years from remission showed 0.2-4.5% m-BCR/ALB(+) cells in the peripheral blood (PB), which is within the accepted background range for this method. The combined analysis found that all the m-BCR/ABL(+) cells were mature lymphocytes. Because mature lymphocytes have a long life span in the circulation, this finding supports the fact that the patient is in remission. Moreover, since mature differentiated cells have a low proliferative capacity, there is a low risk for relapse.     

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.     

Deletion of any part of the BCR or ABL gene on the derivative chromosome 9 is a poor prognostic marker in chronic myelogenous leukemia

To evaluate the prognostic significance of submicroscopic deletions of the ABL or BCR gene associated with t(9;22) in chronic myelogenous leukemia (CML), we investigated the incidence of an ABL or BCR deletion on derivative chromosome 9 using fluorescence in situ hybridization (FISH). FISH was performed using the LSI BCR/ABL dual-fusion translocation probe on bone marrow cells of 86 patients with CML. Of 86 patients, ABL deletion was detected in 13 (15.1%) patients and BCR deletion in 8 patients (9.3%). Patients with ABL deletion showed shorter event-free survival time (EFS) than those without ABL deletion (P = 0.020). Patients with BCR deletion showed significantly short overall survival time (OS; P = 0.039). Patients with ABL and/or BCR deletion (14/86 patients, 16.3%) showed significantly short OS and EFS (median OS, 43.0 months; median EFS, 40.0 months), compared to the patients without any BCR or ABL gene deletions (median OS, 94.0 months; median EFS, 90.0 months; P = 0.041 for OS, P = 0.008 for EFS). All the patients with BCR deletion, except for one, had a concomitant ABL deletion, suggesting that BCR deletion occurs in conjunction with ABL deletion. In patients with ABL deletion only, BCR/ABL rearrangement with b2a2 mRNA type tended to be more frequent than in patients without any deletion of the two genes (P = 0.073). Deletion of any of the BCR or ABL genes on derivative chromosome 9 was associated with both short OS and EFS. We conclude that deletion of not only the ABL gene, but also of the BCR gene, is a poor prognostic marker that indicates rapid disease progression in CML.     

荧光原位杂交技术检测BCR/ABL融合基因的研究

目的建立荧光原位杂交技术平台,应用FISH技术检测在CML检测BCR/ABL融合基因,探讨FISH技术在在CML中应用的价值。方法应用FISH对BCR/ABL探针进行前期的验证,建立正常阈值,再应用该探针检测CML中BCR/ABL融合基因,进行临床检测评估。结果主要假阳性信号模式的正常阈值为1G1R1F 11%、1G1R2F 2%。75例样本FISH检测出48例阳性,15例经细胞遗传学检测,12例检测结果与FISH结果一致,3例CG为阴性,FISH检测为阳性。结论荧光原位杂交技术应用于临床检测之前应进行探针的前期验证,制定一套规范实验流程,且FISH技术在CML诊断、分型、临床治疗方案的制定、预后的判断以及微小残留病变检测上均有重要的价值。     

New highly sensitive fluorescence in situ hybridization method to detect PML/RARA fusion in acute promyelocytic leukemia

We investigated a new fluorescence in situ hybridization (FISH) method to detect PML/RARA fusion and/or anomalies of the RARA gene (alias RARalpha) in interphase nuclei from patients with acute promyelocytic leukemia (APL). This method uses a commercially available product with two different colored fluorescent probes to detect both PML/RARA gene fusion products (double fusion signal or dual-color fluorescence in situ hybridization [D-FISH]). A total of 82 bone marrow specimens were studied, including 30 from normal bone marrow transplant donors, 33 from patients with untreated APL, 14 from patients with treated APL, and 5 from APL patients with known translocation variants or alternate translocations. The signal patterns and percentage of abnormal nuclei were determined in a blinded study on 500 interphase nuclei for each specimen. Based on 25 normal specimens, the normal cutoff was >0.6% and >1.6% for t(15;17) and t(17;var), respectively. The clinical sensitivity for this series of patients was 98% and the clinical specificity was 100%. The results suggest that the new D-FISH probe set can detect all t(15;17)(q22;q21) and all variant forms of this translocation associated with PML and RARA. In addition, this FISH method can detect all alternate translocations involving RARA and not PML. This FISH method can be used both for the accurate diagnosis of APL and to monitor low levels of disease in treated patients.     

Fluorescence in situ hybridization BCR/ABL fusion signal rate in interphase nuclei of healthy volunteer donors: a test study for establishing false positive rate

Fluorescence in situ hybridization (FISH) using chromosome-specific DNA probes is rapidly becoming a part of clinical laboratory practice. However, as a relatively new clinical test, it is not yet standardized and for practical reasons each laboratory must establish its own criteria. For this purpose we have evaluated the specificity of a dual-color BCR/ABL translocation probe by establishing the range of BCR/ABL fusion-positive scores in a healthy donor group. The false positive rate (FPR), determined by the percent of FISH BCR/ABL fusion-positive cells found in the specimens of healthy donors, was estimated at 2.3% (mean = 1%-4%). Thus the cut-off value for false positive nuclei was set at 5%.     

Molecular and cytogenetic characterization of a novel rearrangement involving chromosomes 9, 12, and 17 resulting in ETV6 (TEL) and ABL fusion

We performed chromosome analysis on the bone marrow of a patient with BCR/ABL negative chronic myelogenous leukemia (CML). By interphase fluorescence in situ hybridization (FISH), an extra ABL signal was present in interphase nuclei and appeared to be located at 17p in the metaphase cells. Chromosome analysis showed a subtle abnormality at 17p13 and 12p13 but no visible rearrangement at 9q34 (ABL). Additional FISH experiments disclosed a rearrangement between the short arms of chromosomes 12 and 17 at approximately bands 12p13 and 17p13, respectively. In addition, subtelomeric FISH analysis confirmed the presence of terminal 12p at 17p13 and showed terminal 9q34 to be intact on each chromosome 9. Taken together, these results indicated a rearrangement involving chromosomes 9, 12, and 17 that suggested the possibility of juxtaposition of part of the ETV6 (also known as TEL) locus (12p13) with a portion of ABL (9q34) together at 17p13. The ETV6/ABL fusion was confirmed by RT-PCR, which showed that the first 5 exons of ETV6 were fused in frame with ABL at exon 2. Wild-type ETV6 and ABL were also expressed, in accordance with the FISH results that showed no loss of the second ETV6 or ABL allele.     

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。