BCR/ABL fusion gene detected on 9q34 by fluorescence in situ hybridization in an acute leukemia with two BCR/ABL positive clones, one Ph-negative and one Ph-positive.

We report cytogenetic, fluorescence in situ hybridization (FISH), and molecular analyses in the first reported case of an acute leukemia with two BCR-positive clones: one cell Ph-positive and all others Ph-negative. A BCR/ABL fusion gene on 9q34 was detected only with a BCR/ABL dual color translocation probe. These FISH interphase signals must be confirmed on a metaphase to avoid an erroneous interpretation. This observation appears to indicate a 2-step mechanism for this aberrant fusion gene localization: first, a classical t(9;22), and then the transfer of the fusion gene formed on chromosome 22 to chromosome 9 by a second translocation between the long arms of the derivative chromosomes 9q+ and 22q-, masking the first chromosome exchange.     

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。     

BCR/ABL amplification in chronic myelocytic leukemia blast crisis following imatinib mesylate administration

The onset of accelerated phase or blast crisis of chronic myelocytic leukemia (CML) is usually associated with the acquisition of new chromosome abnormalities in addition to the t(9;22)(q34;q11) that is characteristic of the chronic phase CML. We describe the cytogenetic and molecular genetic findings in two cases of myelocytic blast crisis of CML, one occurring 6 months after commencing treatment with the ABL-specific tyrosine kinase inhibitor imatinib mesylate (STI571, Glivec, or Gleevec) and the second treated with imatinib mesylate for established blast crisis. In both cases, multiple secondary cytogenetic abnormalities were observed at transformation, with homogeneously staining regions that were shown to contain BCR/ABL amplification by fluorescence in situ hybridization appearing after imatinib mesylate administration. BCR/ABL amplification is emerging as an important mechanism of acquired resistance to imatinib mesylate.     

Amplification of the BCR/ABL fusion gene clustered on a masked Philadelphia chromosome in a patient with myeloblastic crisis of chronic myelocytic leukemia

Although the chronic phase of chronic myelocytic leukemia (CML) is characterized by the Philadelphia (Ph) chromosome creating a hybrid BCR/ABL gene, additional genetic changes involved in blast crisis are poorly understood. We report a 4-8-fold amplification by tandem duplication of the BCR/ABL fusion gene clustered on a masked Ph chromosome in a 61-year-old male patient with CML in myeloblastic crisis. Our finding suggests that the BCR/ABL amplification may play a role as a novel mechanism in the progression to an aggressive blast transformation in some cases of Ph-positive CML.     

A fluorescence in situ hybridization study of complex t(9;22) in two chronic myelocytic leukemia cases with a masked Philadelphia chromosome

The t(9;22)(q34;q11) is evident in more than 90% of patients with chronic myelocytic leukemia (CML) and gives rise to the Philadelphia chromosome (Ph). Approximately 5%-10% of CML patients show variant translocations involving other chromosomes in addition to chromosomes 9 and 22. In some variant translocations, additional material is transferred on der(22), resulting in a masked Ph chromosome. In this paper, we report two apparently Ph-negative (Ph-) CML cases showing a t(7;9;22)(q22;q34;q11) and a t(8;9;22)(q12;q34;q11), respectively. A detailed molecular cytogenetic characterization was performed by fluorescence in situ hybridization (FISH), which disclosed the presence of the 5'BCR/3'ABL fusion gene on the der(7) and der(8) chromosomes, respectively. Derivative (22) appeared as a masked Ph chromosome in both cases. FISH analysis with appropriate BAC/PAC clones allowed us to precisely characterize the complex chromosomal rearrangements that were not detected by conventional cytogenetic analysis.     

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。     

应用组合荧光原位杂交技术检测慢性淋巴细胞白血病的基因组异常

目的 探讨应用组合荧光原位杂交(panel fluorescence in situ hybridization，panel FISH)技术对慢性淋巴细胞性白血病(chronic lymphocytic leukaemia，CLL)基因组异常检测的价值。方法 分别应用3号、12号、18号的着丝粒探针和序列探针D13S272(13q14．3)、ATM(11q23)等5种荧光素标记的DNA探针，对22例CLL患者进行FISH检测，并和常规细胞遗传学检测结果进行比较，以确定何种方法对检测CLL的染色体和基因组异常更为敏感可靠。结果 22例CLL患者中，常规细胞遗传学检测出8例(36．3％)有染色体异常，包括单纯 123例； 3、 12l例； 3、 12、 18 1例；t(5；15)1例；13q-1例；3q-、18p 1例；4q 和13q-1例；组合FISH检测出15例(68，1％)有染色体异常，包括 34例、 126例、 18l例、11q-6例、13q-8例。结论组合。FISH技术是检测CLL患者染色体基因组异常的有效手段，与常规细胞遗传学方法相结合则可明显提高CLL染色体异常的检出率。     

Cytogenetic abnormalities detected by fluorescence in situ hybridization on paraffin-embedded chronic lymphocytic leukemia/small lymphocytic lymphoma lymphoid tissue biopsy specimens

Cytogenetic fluorescence in situ hybridization (FISH) panels are a major prognostic tool in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), but few data exist on using paraffin-embedded extramedullary tissue biopsy specimens for these purposes. Isolated whole nuclei were extracted from 20 paraffin-embedded tissue biopsy specimens with CLL/SLL and analyzed using a standard CLL FISH panel. FISH studies were successful in 18 (90%) of 20 cases, and chromosomal abnormalities were detected in 18 (100%) of the technically successful cases. Deletion 13q14.3 was most frequent (10 [56%]; isolated in 8 and with other abnormalities in 2), followed by trisomy 12 (5 [28%]), deletion 11q22.3 (4/16 [25%]), 14q32 (IGH@) translocation (3 [17%]), and deletion 17p13.1 (1/16 [6%]). One case with IGH@ translocation showed a BCL2 translocation partner. No cases showed 6q23 deletion. Results of this FISH panel performed on 42 additional peripheral blood (PB)/bone marrow (BM) CLL specimens were similar except for a significantly greater frequency of deletion 13q14.3 in combination with other aberrations. Cytogenetic FISH studies using paraffin-embedded tissue biopsy specimens in CLL/SLL had a high yield and, with 1 exception, demonstrated a profile similar to cases diagnosed in PB/BM.     

Deletion of 6q27 in chronic lymphocytic leukemia and multiple myeloma detected by fluorescence in situ hybridization.

Nonrandom deletions of the long arm of chromosome 6 (6q) are associated with various lymphoid malignancies. It has been suggested that deletions of 6q25-27, 6q21, and 6q23 typically occur in intermediate-grade, high-grade, and low-grade lymphomas, respectively. We used fluorescence in situ hybridization (FISH) to evaluate the occurrence of 6q27 deletion in chronic lymphatic leukemia (CLL) and multiple myeloma (MM). 6q27 deletion was detected in 21% of patients with CLL and in 28% of patients with MM. The percentage of cells containing deletions ranged between 25-49. Two patients with MM had progressive disease and the aberration was detected in both. We conclude that FISH is a sensitive method to detect 6q27 deletion in lymphoproliferative disorders. Also, this deletion is not specific to intermediate-grade lymphomas, but occurs also in CLL and MM.     

11q trisomy detected by fluorescence in situ hybridization

Takano T, Yamanouchi Y, Kawashima S, Date M, Hashira S, Kida M, Abe T, Nakahori Y, Nakagome Y. 11q trisomy detected by fluorescence in situ hybridization. Clin Genet 1993: 44: 324–328. © Munksgaard, 1993 A patient with psychomotor developmental delay, multiple minor anomalies, congenital heart disease and left inguinal hernia is reported. His karyotype was 45,X/46,X,+mar (3 : 37 cells), and the marker chromosome was identified as t(Y;11) (q12;q14?) using fluorescence in situ hybridization and fluorescent chromosome painting. He was diagnosed as mosaic for de novo 11q trisomy.     

Epididymal sperm aneuploidies in three strains of rats detected by multicolor fluorescence in situ hybridization

A multicolor fluorescence in situ hybridization (FISH) method was developed to detect aneuploidy and diploidy in epididymal sperm of rats using DNA probes specific for chromosomes 4 and Y. Fourteen healthy young-adult rats from three strains were evaluated: inbred Fisher 344/N/ehs, outbred Sprague-Dawley, and outbred WU Wistar/CPB. The hybridization efficiency of the FISH procedure was >99.9%, the sex-ratio in sperm was ∼1 as expected, and there was no significant variation among two independent scorers. No significant variations were detected within or among strains in the frequencies of sperm disomy for chromosome 4 (1–6.5 per 10,000 cells per animal) or the Y chromosome (0–2.5 per 10,000 cells per animal). There was a trend toward increased variation among Wistar rats. The frequencies of sperm-carrying hyper- and hypohaploidy for chromosome 4 were similar, suggesting a symmetrical mechanism of chromosome gain and loss during meiosis. The frequencies of Y-Y-4-4 sperm, which represent genomic meiosis II errors, did not differ significantly across strains (0.1–0.7 per 10,000 cells per strain). This FISH method for detecting aneuploidy in rat epididymal sperm provides a promising interspecies biomarker of male germ cell aneuploidy and introduces the rat as an animal model for investigating the heritable risk to offspring associated with paternal genotype, physiology, and exposure to environmental mutagens. There appear to be no significant differences among young healthy rats, mice, and men in the baseline frequencies of sperm with Y chromosomal disomy, the only chromosome for which data currently exists for all three species. Environ. Mol. Mutagen. 31:125–132, 1998 © 1998 Wiley-Liss, Inc.     

Y chromosome loss in chronic myeloid leukemia detected in both normal and malignant cells by interphase fluorescence in situ hybridization

Loss of the Y chromosome in bone marrow (BM) cells is a normal age-associated event. Y chromosome loss is also observed in the Philadelphia chromosome (Ph) positive BM cells of some patients with chronic myeloid leukemia (CML) in chronic phase, but at a younger age than in normal individuals. While the significance of loss of the sex chromosome in normal males is uncertain, -Y marrow cells are not believed to be of clonal origin. However, because CML is a clonal disease, CML sub-populations with Y loss may constitute a disease-related sub-clone. We used a PCR-amplified yeast artificial chromosome containing the BCR gene region for single color interphase analysis of BCR rearrangement by fluorescence in situ hybridization (FISH). Then, using two color FISH, with one fluorochrome detecting the BCR gene region and the other detecting Y chromosome repeat sequences, we surveyed peripheral and BM Y loss in both normal Ph- (BCR not disrupted) and CML Ph+ (BCR rearranged) interphase nuclei of two patients with Y loss in Ph positive cells observed by metaphase analysis. -Y was seen in a proportion of Ph+ cells in both cases, and the proportion matched that seen in Ph- cells, indicating that Y loss is probably sporadic in both normal and CML populations, and that the propensity for Y loss in normal BM cells may be a phenotype that can be retained by malignant cells in CML.     

应用荧光原位杂交技术检测卵巢浆液性囊腺癌中ZNF217基因拷贝数

目的探讨锌指蛋白217（zinc finger protein 217，ZNF217）基因在卵巢浆液性囊腺癌中20号染色体基因拷贝数改变情况及其临床意义。方法应用荧光原位杂交技术及LSI ZNF217探针对2种卵巢癌细胞株SKOV3、HO-8910和23例卵巢浆液性囊腺癌、10例浆液性囊腺瘤及7份正常卵巢组织进行检测。结果两种卵巢癌细胞株及12例卵巢癌出现ZNF217基因扩增，浆液性囊腺瘤有1例发生了基因的扩增，其余的及正常卵巢组织未出现基因扩增，卵巢癌与卵巢浆液性囊腺瘤、正常卵巢细胞相比较，ZNF217基因拷贝数改变差异有统计学意义（P〈0．05），而低分化的卵巢癌比高分化发生ZNF217基因扩增的几率明显增高（P〈0．05），Ⅲ～Ⅳ期比Ⅰ期的卵巢癌ZNF217基因拷贝数明显增多（P〈0．05）。结论ZNF217基因很可能是卵巢癌发生的促进因子之一，并与卵巢癌分化及预后不良有关。     

Prognostic relevance of monosomy at the 13q14 locus detected by fluorescence in situ hybridization in B-cell chronic lymphocytic leukemia

Deletion of the chromosome band 13q14, which contains the putative deleted in B-cell malignancy (DBM) gene, and trisomy 12 have been demonstrated by fluorescence in situ hybridization (FISH) techniques in malignant B-cells from patients with B cell chronic lymphocytic leukemia (B-CLL). However, the prognostic relevance of 13q14 abnormalities as detected by FISH is unknown. We prospectively studied malignant blood cells from 54 consecutive, untreated B-CLL patients using FISH probes to the RB1 locus and DBM (markers D13S25 and D13S319) for band 13q14, as well as probes to chromosome 12. The cells from all cases were CD5+ CD20+, expressed clonally restricted surface immunoglobulin light chain, and had typical features for B-CLL on careful blood smear morphologic evaluation. Patients were followed for a mean of 3.9 years and treatment-free survival (TFS) was used in the prognostic factor analysis. Twenty-four (44%) patients were observed to have monosomy of the RB1 locus and 26 (48%) monosomy of D13S25 and D13S319. The 26 patients who had a deletion at at least one of these loci had a 48.4 month (mo) median TFS vs 31.1 mo for those without evidence of deletion at any 13q14 locus (p = 0.07). The seven patients found to have trisomy 12 had a median TFS of 6.9 mo vs 39.3 mo for those diploid for chromosome 12 (p < 0.01). When these seven patients with trisomy 12 were excluded from the analysis, patients who had a deletion at 13q14 tended to have a longer median TFS (50.1 vs 36.2 mos), but this was not statistically significant (p = 0.2). This study confirms the prevalence of 13q14 deletions in B-CLL and suggests that patients with this abnormality have a better TFS than those with trisomy 12.     

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.     

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.