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Chronic myelogenous leukemia (CML) is associated with an acquired karyotypic abnormality, the Philadelphia (Ph) chromosome, in 95% of cases. The Ph chromosome is the product of a balanced translocation that results in a hybrid gene that is considered essential for the pathogenesis of this disease. We have found a complex translocation involving chromosomes 9, 12, and 15 in a 42-year-old Haitian male with the clinical findings of CML. Complex translocations have been shown to result in the masking of the Ph chromosome. We used a mixture of two BCR-specific DNA probes for Southern blot analysis in order to test this hypothesis in our patient. High-molecular weight DNA was digested with the restriction enzymes BglII, BamHI and HindIII. The BglII digestion revealed the presence of two abnormal fragments of 3.9 and 3.0 kb and the BamHI digestion an abnormal 15-kb fragment. These data suggest there is a breakpoint in region 2 of M-bcr. The identification of this breakpoint confirms our hypothesis that a rearrangement involving 22q11 has occurred in the leukemic cells of our patient. A secondary translocation involving chromosomes 12 and 15 has hidden the effects of this translocation. Combined cytogenetic and molecular analysis establishes the karyotype of our patient as 46,XY,t(9;12;15;22)(q34;q12;q21;q11).

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.

The Ph chromosome was the first specific karyotype abnormality associated with a particular neoplastic disease in humans. For many years it was suspected that chromosome abnormalities might cause cancer by alteration of specific genes or their expression. Significant recent developments in our understanding of the molecular consequences of the Ph translocation strengthen that assumption. The Ph translocation generates a hybrid gene consisting of 5' regulatory, promotor, and exon sequences of the bcr gene on chromosome 22 fused to 3' exons and polyadenylation/termination sequences of the ABL proto-oncogene from chromosome 9. It is well established that fusion of bcr and abl genes plays a crucial role in the pathogenesis of CML and ALL. Molecular methods can therefore be used as diagnostic tools to detect the Ph chromosome. Presently, the model of oncogenesis provided by our knowledge of how the abl proto-oncogene becomes activated as a result of the Ph translocation is one of the clearest models of oncogene activation. Despite the progress made, many areas remain to be explored. One important question is, how the hybrid protein is involved in leukemia. Research aimed at investigating the normal function of abl and bcr may be important in efforts to understand their abnormal functioning in leukemia and to increase our understanding of the disease.

A number of protooncogenes have been implicated in human tumorigenesis. The ABL oncogene is consistently rearranged and activated as a consequence of the translocation t(9;22) that gives rise to the Philadelphia chromosome in chronic myeloid leukemia and in some cases of acute lymphoblastic leukemia. Here we describe rearrangement of ABL in a different type of malignancy. The glioblastoma cell line A172 lacks germline alleles of ABL. A recombination event, presumably followed by a duplication, has created two ABL alleles in which exon 11 is joined to chromosome 16 sequences. Although the main body of ABL exons was still present, two considerably shortened ABL mRNAs of 3.8 and 2.8 kilobases were detected; the 3.8-kilobase mRNA hybridized exclusively to an exon IB probe. Neither mRNA hybridized to an ABL probe encompassing part of the tyrosine kinase domain. Thus, the cell line A172 is able to survive in the absence of a functional ABL gene product, indicating that the role of ABL is unlikely to be "housekeeping."

The breakpoint regions of both translocation products of the (9;22) Philadelphia translocation of CML patient 83-H84 and their normal chromosome 9 and 22 counterparts have been cloned and analysed. Southern blotting with bcr probes and DNA sequencing revealed that the breaks on chromosome 22 occurred 3' of bcr exon b3 and that the 88 nucleotides between the breakpoints in the chromosome 22 bcr region were deleted. Besides this small deletion of chromosome 22 sequences a large deletion of chromosome 9 sequences (greater than 70 kb) was observed. The chromosome 9 sequences remaining on the 9q+ chromosome (9q+ breakpoint) are located at least 100 kb upstream of the v-abl homologous c-abl exons whereas the translocated chromosome 9 sequences (22q-breakpoint) could be mapped 30 kb upstream of these c-abl sequences. The breakpoints were situated in Alu-repetitive sequences either on chromosome 22 or on chromosome 9, strengthening the hypothesis that Alu-repetitive sequences can be hot spots for recombination.

The Philadelphia (Ph') chromosome, an abnormal chromosome 22 (ref. 1), is one of the best-known examples of a specific human chromosomal abnormality strongly associated with one form of human leukaemia, chronic myelocytic leukaemia (CML). The finding that a small region of chromosome 9 which includes the c-abl oncogene is translocated to chromosome 22 prompted studies to elucidate the molecular mechanisms involved in this disease. We have demonstrated previously that the chromosome 9 of one patient with CML contains a breakpoint 14 kilobases (kb) 5' of the most 5' v-abl-homologous exon. These data suggest a role for c-abl in CML, a theory supported by the presence of an abnormally sized abl messenger RNA and protein in the CML cell line K562. The region involved in the translocation on chromosome 22 has also been identified: all Ph'-positive patients examined to date have a breakpoint within a 5.8-kb region, for which we have proposed the name 'breakpoint cluster region' (bcr). To determine whether bcr contains protein-encoding regions, probes from bcr were tested for their ability to hybridize to complementary DNA sequences. A 0.6-kb HindIII/BamHI bcr restriction enzyme fragment proved suitable for isolating several cDNA clones from a human fibroblast cDNA library. Using bcr cDNA sequences, we obtained data strongly suggesting the presence of a chimaeric bcr/abl mRNA in the leukaemic cells of Ph'-positive CML patients. The recent isolation of cDNA clones containing bcr and abl sequences confirms this finding. Because the bcr part of the chimaeric mRNA could be required to induce the transforming activity of the human c-abl oncogene, we have now initiated studies to characterize the normal 'bcr gene' and to determine the effect of a translocation within its coding domain. We demonstrate that as a result of the Ph' translocation, a variable number of bcr exons are included in the chimaeric bcr/abl mRNA. The bcr gene sequences in this mRNA could be responsible for the transition of the abl cellular proto-oncogene into an oncogene.

We have identified and molecularly cloned 46 kb of human DNA from chromosome 22 using a probe specific for the Philadelphia (Ph') translocation breakpoint domain of one chronic myelocytic leukemia (CML) patient. The DNAs of 19 CML patients were examined for rearrangements on chromosome 22 with probes isolated from this cloned region. In 17 patients, chromosomal breakpoints were found within a limited region of up to 5.8 kb, for which we propose the term "breakpoint cluster region" (bcr). The two patients having no rearrangements within bcr lacked the Ph' chromosome. The highly specific presence of a chromosomal breakpoint within bcr in Ph'-positive CML patients strongly suggests the involvement of bcr in this type of leukemia.

Somatic cell hybrids have been constructed between a thymidine kinase-deficient mouse cell line and blood leukocytes from a patient with acute promyelocytic leukemia showing the 15q+;17q- chromosome translocation frequently associated with this disease. One hybrid contains the 15q+ translocation chromosome and very little other human material. We have shown that the c-fes oncogene, which has been mapped to chromosome 15, is not present in this hybrid and, therefore, probably is translocated to the 17q- chromosome. Analysis of the genetic markers present in this hybrid has enabled a more precise localization of the translocation breakpoints on chromosomes 15 and 17. Our experiments also have enabled an ordering and more precise mapping of several genetic markers on chromosomes 15 and 17.

The Philadelphia (Ph) chromosome, the product of t(9:22), is the cytogenetic hallmark of chronic myelogenous leukemia. The c-abl oncogene on chromosome 9 is translocated to the Ph chromosome and linked to a breakpoint cluster region (bcr), which is part of a large bcr gene. This results in the formation of a bcr-c-abl fusion gene, which is transcribed into an 8.5 kb chimeric mRNA encoding a 210 kd bcr-c-abl fusion protein. The Ph chromosome is also found in acute lymphoblastic leukemia (Ph+ ALL). Although the c-abl is translocated and a new 190 kd c-abl protein has been identified, no breakpoints are observed in the bcr (Ph+bcr- ALL). Here we show that in Ph+bcr- ALL, breakpoints in chromosome 22 occur within the same bcr gene, but more 5' of the bcr. Cloning of a chimeric bcr-c-abl cDNA demonstrates that the fusion gene is transcribed into a 7 kb mRNA, encoding a novel fusion protein.

By analysis of a series of somatic cell hybrids derived by fusion of either mouse or Chinese hamster cells with leukocytes from different chronic myelocytic leukemia (CML) patients or from normal donors, we have localized the human oncogene, c-sis, on the q11 to qter segment of chromosome 22 and demonstrated its translocation from chromosome 22 to chromosome 9 (q34) in CML.

Chromosomal analysis of a patient with chronic myelocytic leukemia (CML) revealed a translocation (9;12) (q34;q21) without a detectable Philadelphia chromosome (Ph1). Using molecular approaches we demonstrate (i) a rearrangement within the CML breakpoint cluster region (bcr) on chromosome 22, and (ii) a joint translocation of bcr and c-abl oncogene sequences to the derivative chromosome 12. These observations support the view that sequences residing on both chromosome 9 (c-abl) and 22 (bcr) are involved in the generation of CML and suggest that a subset of Ph1-negative patients may in fact belong to the clinical entity of Ph1-positive CML.

The hallmark of chronic myelogenous leukemia (CML) is a translocation between chromosomes 9 and 22 - the Philadelphia (Ph') translocation. The translocation is also found in acute lymphocytic leukemia (ALL) albeit in a lower percentage of patients. The breakpoint on chromosome 22 is located within the BCR gene: in CML, breakpoints are clustered within 5.8 kb of DNA, the major breakpoint cluster region (Mbcr). In ALL, breakpoints have been reported within the Mbcr but also in more 5' regions encompassing the BCR gene. To characterize the latter breakpoints, we have molecularly cloned and mapped the entire gene, which encompasses approximately 130 kb of DNA. Mbcr negative, Ph'-positive ALL breakpoints were not distributed at random within the gene but rather were found exclusively within the 3' half of the first BCR gene intron. In contrast to the Mbcr, which is limited to a region of 5.8 kb, this part of the intron has a size of 35 kb. Translocation breakpoints in this region appear to be specific for ALL, since it was not rearranged in clinically well-defined CML specimens nor in any other tumor DNA samples examined.

Breakpoints on chromosome 22 in the translocation t(9;22) found in Philadelphia positive acute lymphoblastic leukaemia patients fall within two categories. In the first the breakpoint is localized within the breakpoint cluster region of the BCR gene, analogous to the chromosome 22 breakpoint in chronic myeloid leukaemia. The second category has a breakpoint 5' of this area, but still within the BCR gene. We have previously shown that these breakpoints occur within the first intron of the BCR gene and cloned the 9q+ junction from such a patient. We have now determined the sequences around the breakpoints on both translocation partners from this patient as well as the germline regions. The chromosome 9 ABL sequence around the breakpoint shows homology to the consensus Alu sequence whereas the chromosome 22 BCR sequence does not. At the junction there is a 6 bp duplication of the chromosome 22 sequence which is present both in the 9q+ and in the 22q- translocation products. Possible mechanisms for the generation of the translocation are discussed.