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.     

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.     

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.     

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.     

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.     

Multiple genetic events involving rbi gene deletion and amplification of chromosome 21 in a case of acute lymphocytic leukemia

A patient with acute lymphocytic leukemia was found to have a hyperdiploid karyotype, characterized by hexasomy 21 and del(7)(p15). It was shown that the four extra copies of chromosome 21 were all derived from only one of the homologous chromosomes. Molecular analysis showed that the patient had a deletion of both alleles of the retinoblastoma (RBI) gene. These results suggest that multiple events, including loss of RBI gene function and amplification of a key gene(s) on chromosome 21, have contributed to the leukemic transformation. Genes Chrom Cancer 9:72-75 (1994). © 1994 Wiley-Liss, Inc.     

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.     

Cytogenetic and molecular characterization of double inversion 3 associated with a cryptic BCR-ABL1 rearrangement and additional genetic changes

Rearrangements of chromosome 3 involving bands 3q21 and 3q26 have been reported in about 2% of patients with acute myeloid leukemia, and rarely in myelodysplastic syndrome or chronic myelogenous leukemia (CML). To date, only six cases of inversion of both homologues have been reported. Loss of normal chromosome 3 and duplication of the inverted chromosome have been proposed as the most likely mechanism, but have not been shown experimentally. We present a 36-year-old male with an initial diagnosis of CML and resistance to imatinib mesylate. Chromosome analysis showed an inversion within the long arm of both homologues of chromosome 3 and an interstitial deletion within the long arm of one chromosome 7. The rearrangement of EVI1 locus on both homologues of chromosome 3 was confirmed by fluorescence in situ hybridization (FISH). Additional FISH studies showed a cryptic insertion of ABL1 into the BCR region, and subsequent duplication of the derivative chromosome 22. The single-nucleotide polymorphism array showed copy-neutral loss of heterozygosity on chromosomes 3 and 22, suggesting that a somatic repair mechanism is involved in the evolution of these genetic alterations. This case illustrates the complexity of genetic aberrations in neoplastic cells, and the value of array technology, used in concert with conventional cytogenetic methods, for a better understanding of the pathogenesis.     

A complex chromosome rearrangement forms the BCR-ABL fusion gene in leukemic cells with a normal karyotype

Chromosome in situ hybridization studies showed that the normal karyotype of leukemic cells from a patient with Ph1-negative, BCR-positive chronic myeloid leukemia (CML) concealed a complex t(9;22;20)(q34;q11;p13). The close association of 5'-BCR and 3'-ABL was demonstrated by field inversion gel electrophoresis, and in situ hybridization showed that BCR-ABL was located on the short arm of chromosome 20. Our findings further indicate that chromosome rearrangement is the cause of BCR-ABL gene fusion in leukemic cells that show a normal karyotype. Results from in situ hybridization studies were consistent with formation of the t(9;22;20) by a two step chromosomal rearrangement, but field inversion gel electrophoresis results indicated a more complex rearrangement.     

Chromosome microdissection in leukemia: A powerful tool for the analysis of complex chromosomal rearrangements

In many human cancers the presence of marker chromosomes or unbalanced translocations prevents complete karyotypic analysis. Chromosome microdissection has become an increasingly important method for assessing chromosome rearrangements. However, most studies using chromosome microdissection have been carried out on established cancer cell lines that provide an unlimited supply of abnormal metaphase cells. We have routinely performed microdissection of as few as three marker chromosome copies from short-term cultures of acute myeloid leukemias, followed by in vitro DNA amplification and fluorescence in situ hybridization (FISH) to normal metaphase spreads. Using this type of “reverse chromosome painting,” we were able to characterize precisely the chromosomal constitution of each marker chromosome in the samples, confirming the diagnostic usefulness of microdissection in cancer cytogenetics. In addition, in one leukemia with atypical cytological features, microdissection enabled us to detect a novel retinoic acid receptor α gene rearrangement. Genes Chromosom Cancer 15:26–33 (1996). © 1996 Wiley-Liss, Inc.     

Localization of amplified MYC gene sequences to double minute chromosomes in acute myelogenous leukemia

Cytogenetic and molecular studies were performed on two dmin-bearing acute myelogenous leukemia (FAB-M2) samples. Both cases were characterized by complex karyotypes containing interstitial deletions of the long arm of chromosome 8 altering band 8q24.1, aberrations affecting the short arm of chromosome 17, and multiple double minute chromosomes (dmin). Using a 1.4 kb cDNA probe coding for the third exon of the MYC oncogene, DNA slot blots indicated MYC gene sequences were amplified in both samples. Fluorescence in situ hybridization using a 9.0 kb genomic probe for MYC was performed in one we and localized the amplified MYC gene sequences to the dmin. Neither patient achieved a complete remission using traditional induction chemotherapy. The complex karyology with amplification of MYC gene sequences appears to represent a poor prognostic subgroup of acute myelogenous leukemia. Genes Chrom Cancer 9:62-67 (1994). © 1994 Wiley-Liss, Inc.     

Therapy‐related B‐lymphoblastic leukemia associated with Philadelphia chromosome and MLL rearrangement: Single institution experience and the review of the literature

Therapy related acute lymphoblastic leukemia (t‐ALL) of B cell origin is rare and constitutes approximately 2% of all ALL. Previously compiled data on the complete cytogenetic analysis of 48 t‐B‐ALL cases suggested that MLL rearrangement at 11q23 gene locus is the most common abnormality. Philadelphia chromosome (Ph) and a normal karyotype were reported as the second and third most common karyotypes, respectively. We investigated cytogenetic karyotypes of six t‐B‐ALL cases with a pre‐B cell immunophenotype. Ph + t‐B‐ALL was noted in four of six patients previously treated with radiation and/or chemotherapy. In addition, one case demonstrated MLL rearrangement at 11q23 locus while one case demonstrated normal cytogenetic karyotype. Five of the six t‐B‐ALL patients had persistent leukemia following initiation of chemotherapy for secondary leukemia with survival ranging from 10 to 21 months. To our knowledge, only fourteen patients with Ph + t‐B‐ALL have been described in the literature. In the current study, three of four cases with Ph + t‐B‐ALL were associated with treated breast carcinoma while one patient was treated for Hodgkin lymphoma. All four patients had undergone radiation therapy. The results may indicate a plausible association between Ph+t‐B‐ALL and prior radiation exposure.