9q34 rearrangements in BCR/ABL fusion-negative acute lymphoblastic leukemia

The t(9;22)(q11.2;q34) translocation is found in a subset of acute lymphoblastic leukemia (ALL). The presence of this translocation involving the fusion of BCR/ABL genes represents a poor prognostic group. Because of the importance in detecting t(9;22) in ALL patients and because occasionally a cytogenetically cryptic BCR/ABL fusion is detected with fluorescence in situ hybridization (FISH), our laboratory routinely performs BCR/ABL FISH tests on all newly diagnosed ALL patients. In the past year, 25 consecutive, newly diagnosed, untreated ALL cases were analyzed. We report the cytogenetics and FISH findings of three cases containing a rearranged 9q34 region with an intact BCR (22q11.2) region and an absence of the BCR/ABL fusion. A split ABL signal representing a translocation of the 9q34 region with chromosome segments other than 22q11.2 (BCR) was observed in 3 cases. Two of these patients were 3 years old; one was 21 at the time of diagnosis. A split ABL FISH signal without the involvement of BCR does not represent a t(9;22) translocation, and prognostic implications of this apparent subgroup of ALL cases have not been determined. Cytogenetic, pathologic, and clinical aspects of these three cases are presented.     

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。     

Location of the BCR-ABL fusion gene on the 9q34 band in two cases of Ph-positive chronic myeloid leukemia

Two new variant Philadelphia (Ph) chromosomes with an aberrant location of the BCR-ABL fusion gene on 9q34 of the derivative 9 are reported. One presented cytogenetically as a standard t(9;22)(q34;q11), whereas the other was classified as an ins(9;22)(q34;q11.1q11.2) using the combined interpretation of cytogenetic, FISH, and molecular data. The mechanisms of the two rearrangements are presented. It is suggested that the insertion has occurred in a single event in the patient with ins(9;22). In the patient with t(9;22), both a translocation and an insertion, occurring either sequentially or simultaneously, can account for the location of the BCR-ABL fusion gene on the derivative 9. A possible poor prognostic impact of this aberrant location of the BCR-ABL is also suggested by the clinical data reported in such patients. Genes Chromosomes Cancer 20:148–154, 1997. © 1997 Wiley-Liss, Inc.     

Clinical implications of fluorescence in situ hybridization analysis in 13 chronic myeloid leukemia cases: Ph-negative and variant Ph-positive.

Thirteen chronic myeloid leukemia (CML) patients, 10 with variant Philadelphia (Ph) translocations and 3 Ph negative cases, were analyzed by fluorescence in situ hybridization (FISH) with the use of BCR and ABL cosmid probes and a chromosome 22 painting probe. In the variant Ph translocations, the BCR-ABL fusion gene was located on the Ph chromosome; in 1 CML Ph-negative patient, the BCR-ABL fusion gene was located on the Ph chromosome; and, in 2 patients, it was located on chromosome 9. The chromosome 22 painting probe was detected on the third-party chromosome of the variant translocation, and in none of the variant translocations was there any detectable signal on chromosome 9. In CML patients with clonal evolution of a simple Ph, a signal of the chromosome 22 painting probe was detected on the der(9) of the Ph translocation. It was concluded that the variant Ph translocations evolved simultaneously in a three-way rearrangement. The clinical parameters of the 13 patients were similar to those of a large group of CML patients with a simple Ph translocation. It is suggested that, to determine the prognosis of CML patients with a complex karyotype, FISH analysis with a chromosome 22 painting probe be performed.     

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.     

Development of acute promyelocytic leukemia with isochromosome 17q after BCR/ABL positive chronic myeloid leukemia

We describe a pediatric case of acute promyelocytic leukemia with an i(17q) after treatment of BCR/ABL positive chronic myeloid leukemia (CML) for 3.5 years. The patient was treated with Busulphan, alpha-2a interferon, hydroxyurea, and cytosine arabinoside at various times in the course of the chronic phase of CML, because he had no HLA-identical donor for bone marrow transplantation. Hematologic remission was achieved for a short time, but cytogenetic remission was never possible. When promyelocytic blast crisis was diagnosed according to the French-American-British classification, cytogenetic studies revealed an i(17q) as a new feature in our patient. The promyelocytic transformation was associated with the appearance of an i(17q) preceding CML are discussed in the light of recent literature.     

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.     

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.     

Trisomy 9 in a patient with secondary acute myelogenous leukemia detected by fluorescent in situ hybridization.

Fluorescent in situ hybridization (FISH) is a molecular cytogenetic technique that is playing an increasingly important role for augmenting the findings of conventional cytogenetics. Here we present the case history of a patient with the clinical diagnosis of secondary acute myelogenous leukemia whose bone marrow cells were found to be hyperdiploid with an extra C group chromosome in a less than optimal preparation. By using FISH the extra chromosome was unequivocally determined to be a chromosome 9. The detection of trisomy 9 in this patient underscores the utility of FISH as an adjunct to GTG banding in the routine diagnosis and management of leukemic patients.     

Translocation of BCR to chromosome 9: A new cytogenetic variant detected by FISH in two Ph-negative,BCR-positive patients with chronic myeloid leukemia

Leukemic cells from two patients with Philadelphia-negative chronic myeloid leukemia (CML) were investigated: I) Cytogenetics showed a normal 46.XY karyotype in both cases, 2) molecular studies revealed rearrangement of the M-BCR region and formation of BCR-ABL fusion mRNA with b2a2 (patient I) or b3a2 (patient 2) configuration, and 3) fluorescence in situ hybridization (FISH) demonstrated relocation of the 5′ BCR sequences from one chromosome 22 to one chromosome 9. The ABL probe hybridized to both chromosomes 9 at band q34, while two other probes which map centromeric and telomeric of BCR on 22q 11 hybridized solely with chromosome 22. For the first time, a BCR-ABL rearrangement is shown to take place on 9q34 instead of in the usual location on 22q 11. A rearrangement in the latter site is found in all Ph-positive CML and in almost all investigated CML with variant Ph or Ph-negative, BCR-positive cases. The few aberrant chromosomal localizations of BCR-ABL recombinant genes found previously were apparently the result of complex and successive changes. Furthermore in patient 2, both chromosomes 9 showed positive FISH signals with both ABL and BCR probes. Restriction fragment length polymorphism (RFLP) analysis indicated that mitotic recombination had occurred on the long arm of chromosome 9 and that the rearranged chromosome 9 was of paternal origin. The leukemic cells of this patient showed a duplication of the BCR-ABL gene, analogous to duplication of the Ph chromosome in classic CML. In addition they had lost the maternal alleles of the 9q34 chromosomal region. The lymphocytes of patient 2 carried the maternal chromosome 9 alleles and were Ph-negative as evidenced by RFLP and FISH analyses, respectively. © 1993 Wiley-Liss, Inc.     

A Ph-negative chronic myeloid leukemia with a complex BCR/ABL rearrangement and a t(6;9)(p21;q34.1)

Chronic myeloid leukemia (CML) is a clonal malignant disorder of a pluripotent hematopoetic stem cell characterized by the presence of the Philadelphia (Ph) chromosome in more than 90% of patients. Cryptic or "masked" BCR/ABL gene rearrangements may be found in cases with a normal karyotype and in cases with the complex karyotype, in which typical t(9;22) is not visible at the microscopic level. Those rearrangements can now be detected by fluorescence in situ hybridization. Here, we report on a novel and complex Ph chromosome-negative CML case with a t(6;9)(p21;q34.1) in which the BCR/ABL fusion gene is located at 6p21.     

Philadelphia-like translocation t(9;22)(q34;q11) found in a follicular lymphoma involving not BCR and ABL but IGL-mediated rearrangement of an unknown gene on 9q34

In a case of follicular center cell lymphoma (FCCL) without evidence of histologic progression towards a high-grade lymphoma, t(9;22)(q34;q11) was found simultaneously with a t(14;18)(q32;q21) and a t(8;14)(q24;q32). Molecular studies of this case showed BCL2 and MYC rearrangements in addition to the rearrangements of immunoglobulin heavy (IGH) and lambda (IGL) loci. Investigation of the t(9;22) using Southern blot and RT-PCR analysis failed to detect M-bcr or m-bcr rearrangements of BCR. Two-color fluorescence in situ hybridization (FISH) with ABL and BCR probes revealed presence of a “fusion” signal, but its atypical localization [der(9)] and gene order [cen-ABL-BCR-tel] indicated that this translocation differed from the t(9;22) in chronic myeloid leukemia and did not involve either ABL or BCR. In addition, further FISH analysis using 9q34- and 22q11-specific probes localized the breakpoint on chromosome 9 distal to the NOTCH1 gene and the breakpoint on 22q11 in the IGL gene cluster. These results indicate an IGL-mediated rearrangement of an unknown gene at 9q34 that together with BCL2 and MYC might be involved in the lymphomagenesis of the present case of FCCL and perhaps in other cases of non-Hodgkin lymphoma in which t(9;22) is sporadically occurring. Genes Chromosomes Cancer 20:113–119, 1997. © 1997 Wiley-Liss, Inc.     

BCR/ABL fusion located on chromosome 9 in chronic myeloid leukemia with a masked Ph chromosome

A reciprocal translocation, t(10; 22) (q22; q11), resulting in a masked Ph chromosome was identified in a patient diagnosed with chronic myeloid leukemia (CML). Both homologs of chromosome 9 were of the normal pattern. Two signals for the ABL probe, both of them hybridized to chromosome 9, were demonstrated via fluorescence in situ hybridization (FISH). Furthermore, cohybridization with two differently labeled BCR/ABL translocation DNA probes indicated a BCR/ABL fusion apparently located on 9q34. Molecular studies revealed a rearrangement of the BCR region and expression of a chimeric BCR/ABL mRNA of CML configuration. These findings indicate that the BCR/ABL fusion resulted from an unusual relocation of the BCR gene from its normal position on 22ql I to 9q34 adjacent to the ABL gene.     

Clinical implications of der(9q) deletions detected through dual-fusion fluorescence in situ hybridization in patients with chronic myeloid leukemia

Poor outcomes of some chronic myeloid leukemia ((CML) patients have been associated with submicroscopic der(9q) deletions, particularly the 5'ABL region. Deletion profiles of 120 BCR/ABL+ CML patients were studied using the dual-fusion fluorescence in situ hybridization probe. Poor prognosis was associated with 5'ABL deletion but not with 3'BCR deletion. Overall survival (OS) and chronic phase duration (CPD) were significantly shorter for 5'ABL deletion than for those without deletions (OS time: 27 vs. 61 months, P = 0.02; CPD: 17 vs. 56 months, P = 0.02). In addition, when isolated 5'ABL deletion patients were compared to those without it, a greater impact on prognosis was detected (OS time: 18 vs. 59 months, P = 0.0008; CPD: 7 vs. 54 months, P = 0.0003). Isolated 5'ABL deletion seems to have a greater impact on survival than does concomitant 5'ABL and 3'BCR deletion, although the difference was not statistically significant in this aspect (OS time: 18 vs. 28 months, P = 0.08).     

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%.     

二氢青蒿素对K562细胞BCR/ABL融合基因表达的影响及意义

目的 探讨二氢青蒿素( dihydroartemisinin,DHA)对白血病细胞K562 BCR/ABL融合基因表达的影响及意义.方法 采用噻唑蓝比色方法检测不同浓度的DHA对于K562细胞的增殖抑制作用,并计算不同培养时间点的抑制率.用逆转录PCR检测K562细胞处理前后BCR/ABL融合基因的表达变化,流式细胞仪检测K562细胞的凋亡情况.结果 DHA(10～160 μmol/L)能抑制K562细胞的增殖,随着浓度增加抑制作用明显增强,培养24 h后抑制率从52.76％增加到94.65％;用浓度为20μmol/L的DHA对K562细胞作用12～48 h后,逆转录PCR检测BCR/ABL融合基因表达,△Ct值为4.45±0.25和5.23±0.21.与对照组(4.23±0.21)相比,差异有统计学意义(P＜0.05).结论 DHA可通过影响BCR/ABL融合基因的表达抑制K562细胞的增殖.这可能是导致K562细胞凋亡的原因之一.     

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.