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.     

Simultaneous occurrence of t(9;22)(q34;q11.2) and t(16;16)(p13;q22) in a patient with chronic myeloid leukemia in blastic phase

Coexistence of two specific chromosomal translocations in the same clone is an infrequent phenomenon and has only rarely been reported in hematological malignancies. We report a combination of t(16;16)(p13;q22), the Philadelphia translocation t(9;22)(q34;q11.2), and deletion of the long arm of chromosome 7 in a patient with chronic myeloid leukemia in blast phase. Monotherapy treatment with imatinib mesylate resulted in the disappearance of the Ph-positive clone, but with persistence of t(16;16) and del(7) in all of the metaphases examined. The case illustrates that, although imatinib mesylate can be an effective treatment in eradication of the BCR–ABL fusion gene cells, the occurrence of additional specific abnormalities in Philadelphia-positive leukemias may pose a significant therapeutic challenge.     

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.     

The second ETV6 allele is not necessarily deleted in acute leukemias with a ETV6/ABL fusion

The ETV6 (TEL) locus at chromosome band 12p13 is a major site of translocations in acute leukemia, particularly in childhood acute lymphoblastic leukemia (ALL). In cases with translocations involving ETV6, the normal ETV6 allele is often deleted. In addition, loss of heterozygosity of ETV6 is frequently observed in childhood ALL. Thus, it has been suggested that ETV6 may have an anti-oncogenic role to play, in addition to its oncogenic role. We have described an unusual case of ALL in which ETV6 is found fused to the ABL gene; ABL is normally activated by fusion to the BCRgene in the 9:22 translocation. We expanded the primary cells from this ETV6/ABLrearranged case of ALL in SCID animals and analyzed them for expression of both ETV6/ABL and the normal ETV6mRNA. We found that both the rearranged and normal ETV6 mRNAs are expressed in the expanded cell population. Furthermore, sequence analysis of the ETV6 PCR product revealed no point mutations which would influence the amino acid sequence. Thus, deletion of the second ETV6 allele is not necessary for the transformation to leukemia by ETV6/ABL. Genes Chromosomes Cancer 21:256–259, 1998. © 1998 Wiley-Liss, Inc.     

Genetic analysis of chromosome 13 deletions in BCR/ABL negative chronic myeloproliferative disorders

Chromosomal deletions of band 13q14 occur recurrently in BCR/ABL negative chronic myeloproliferative disorders (CMPD), including myelosclerosis with myeloid metaplasia (MMM), polycythemia vera (PV), essential thrombocythemia (ET), juvenile chronic myeloid leukemia (JCML), and the so-called BCR/ABL ^? chronic myeloid leukemia (CML). The RB1 tumor suppressor locus, mapping to 13q14, has long since been hypothesized as the important gene. In this report, we have determined the frequency of 13q14 deletions at the molecular level in a large panel of BCR/ABL ^? CMPD at different disease stages and performed a detailed genetic analysis of gross rearrangements/deletions and point mutations of the RBI gene in these disorders. Our data show that molecular deletions of 13q14 are detected in a relatively large fraction of BCR/ABL ^? CMPD (38%), that they appear to be more frequent in MMM than in other BCR/ABL ^? CMPD, and that they may be present at diagnosis or occur during blastic evolution of the neoplasia. The RBI gene displayed a germline configuration in all BCR/ABL ^? CMPD tested, suggesting that 13q14 deletions in these disorders affect a tumor suppressor locus distinct from RB1.     

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.     

ABL amplification in a patient with lymphoid blast crisis of chronic myelogenous leukaemia

 Although chronic phase myelogenous leukaemia (CML) is characterised by the Philadelphia (Ph) chromosome leading to a fusion of the BCR and ABL genes, additional genetic alterations involved in blast crisis are poorly understood. We report an at least 15-fold amplification of the ABL oncogene in a 29-year-old male patient with a variant Ph-positive t(19;22)(p13;q11.2) CML who presented in lymphoid blast crisis. Our finding suggests that an amplification of the ABL oncogene might play a part in the appearance of an aggressive phenotype in some cases of CML. Received: 27 July 1998 / Accepted: 26 October 1998     

Patho-anatomical features of so-called Ph1− chronic myeloid leukemia

Summary Chronic myeloid leukemia without the Philadelphia chromosome (Ph^1– CML) is described and distinguished from chronic myeloid leukemia with the Philadelphia chromosome (Ph¹⁺ CML) on the basis of clinical and autopsy findings of four cases. Ph^1– CML showed clinical, hematological, and patho-anatomical features which could be regarded as typical.Patho-anatomically Ph^1– CML differed from Ph¹⁺ CML in the variable maturation of the leukemic proliferation in the bone marrow and extramedullary infiltrates. Up to the terminal phase Ph^1– CML can be of an extremely mature cell type. However, it can also show myeloblastic transformation after an initially mature cell stage. Ph^1– CML infiltrates are found in tissues and organs which Ph¹⁺ CML usually does not infiltrate or only to a low degree until a blastie crisis.On the basis of its course and clinical and patho-anatomical features Ph^1– CML looks like an atypical chronic myeloid leukemia. However, it is better called an acute myeloid leukemia of the mature cell type.The authors are grateful to Mr. K.-H. Tedsen for his excellent technical assistance and to Mrs. M. Soehring for translation and secretarial help.     

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.     

Rhinovirus identification by BglI digestion of picornavirus RT-PCR amplicons

Rhinoviruses are the main cause of the common cold and precipitate the majority of asthma exacerbations. RT-PCR followed by internal probe hybridisation or Southern blotting, or nested PCRs are currently the most sensitive methods for their identification. However, none of the published techniques can differentiate satisfactorily rhinoviruses from other picornaviruses. Examination of the restriction maps of sequenced rhinoviruses, revealed a highly conserved BglI restriction site (GCCnnnnnGGC), located exactly in the middle of the 380-bp amplicon generated with the OL26–OL27 primer pair, which has been used extensively in the past to identify picornaviruses. Such a site was either not present, or positioned differently in other picornaviruses of known sequence. It was, therefore, considered that digestion of rhinovirus amplicons with this enzyme would result in two equal length fragments, generating a single 190-bp band in gel electrophoresis. In contrast, either one undigested 380-bp band or a double-band pattern would appear in amplicons from other picornaviruses. To test this hypothesis, Bgl digestions of OL26–OL27 amplicons from cultured and wild-type rhinoviruses, whose identity was confirmed by acid lability, as well as from echo, polio and coxsackie viruses were carried out. All rhinovirus samples were digested successfully generating single bands. Among the other picornaviruses, only 6.6% presented a single band pattern, while the rest were as predicted from the model. With a sensitivity of 100% and a specificity over 90%, the method described, which is rapid and remarkably easy to perform, can be used to distinguish rhinoviruses from other picornaviruses to a considerable extent.     

Type 2 Gaucher Disease with Hydrops Fetalis in an Ashkenazi Jewish Family Resulting from a Novel Recombinant Allele and a Rare Splice Junction Mutation in the Glucocerebrosidase Locus

Gaucher disease, the deficiency of the lysosomal enzyme glucocerebrosidase (EC 3.2.1.45), is frequently encountered in the Ashkenazi Jewish population. Carrier screening for Gaucher disease by enzyme analysis performed during a routine pregnancy indicated that both Ashkenazi parents were carriers. Screening for four common Gaucher mutations was subsequently performed on fetal and parental DNA. None of the common Ashkenazi mutations were identified. However, when exons 9–11 were amplified and digested withNciI to detect the L444P mutation, it appeared that the mother and the fetus had an unusual allele and that the expected paternal allele was not present. When the fetal amniocytes were found to have less than 2% of the normal glucocerebrosidase activity and a fetal sonogram revealed hydrops fetalis, the pregnancy was terminated. The diagnosis of severe type 2 Gaucher disease was confirmed at autopsy. Ultrastructural studies of epidermis from the fetus revealed the characteristic disruption of lamellar bilayers, diagnostic for type 2 Gaucher disease. In subsequent studies of the fetal DNA, long-template polymerase chain reaction amplification revealed one appropriately sized band (6.5 kb) and one smaller (5.2 kb) band. Sequencing of the 5.2-kb fragment identified a novel fusion allele resulting from recombination between the glucocerebrosidase gene and its pseudogene beginning in intron 3. This fusion allele was inherited from the father. The result was confirmed by Southern blot analysis using the enzymeSstII. Sequencing of the 6.5-kb fragment identified a previously described, although rare, T-to-G splice junction mutation in intron 10 of the maternal allele, which introduced anNciI site. The couple had a subsequent pregnancy which was also found to be affected. This case study identifies a novel recombinant allele and an unusual splice junction mutation, and demonstrates that even in the Ashkenazi population, screening for common mutations may not accurately identify the most severe forms of the disease.