Detection and localization of breakpoints within the first intron of the BCR gene in Ph1-positive leukemias

We studied breakpoints within the first intron of the BCR gene in 28 Philadelphia (Ph1)-positive acute leukemias (AL) and one Ph1-positive chronic myelogenous leukemia (CML), which lacked rearrangement of major breakpoint cluster region. With a series of genomic probes from this intronic region, we have detected chromosomal breaks in 19 of 28 patients with Ph1-positive AL and one patient with CML. Breakpoints were all located within 30 kb region at the 3' portion of the intron, same as in the previously reported cases. Breakpoints in our cases were not limited within "bcr-2" or "bcr-3", proposed by Chen, et al., but, occurred within or near Alu sequences. Our findings suggest that breakpoints are not randomly distributed throughout this intron, but, there may be some specific sequences that facilitate the process of chromosomal translocation.     

Consistent involvement of the bcr gene by 9;22 breakpoints in pediatric acute leukemias

To investigate the relationship of bcr-abl fusion mRNAs with childhood acute lymphoblastic leukemias (ALL), we examined 27 pediatric Philadelphia chromosome (Ph1)-positive acute leukemias using a reverse polymerase chain reaction (PCR) procedure. In cells from 24 leukemias, single bcr-abl PCR products were detected that corresponded to breakpoints in the minor breakpoint cluster region (mbcr in intron 1 of the bcr gene) associated with production of the P190 fusion protein. Cells from the three remaining leukemias contained breakpoints in the major breakpoint cluster region (Mbcr) as shown by PCR and Southern blot analyses. These three leukemias also contained low levels of the mbcr PCR product that may have resulted from alternative splicing of the bcr-abl precursor RNA. A screen of 35 additional leukemias from patients who failed therapy before day 180 (induction failures or early relapses) found one case with unsuccessful cytogenetics to express Mbcr-abl RNA. All four children with Mbcr breakpoints had white blood cell levels in excess of 250,000 at presentation (compared with 2 of 24 with mbcr breakpoints) and two had hematologic and clinical features suggestive of chronic myelogenous leukemias (CML) in lymphoid blast crisis. Our results indicate that in Ph1-positive pediatric leukemias, all 9;22 breakpoints occur in one of the two known breakpoint cluster regions in the bcr gene on chromosome 22. The reverse PCR reliably detected all patients with cytogenetic t(9;22) and is capable of detecting additional Ph1-positive leukemias that are missed by standard cytogenetics. Furthermore, the Mbcr-type breakpoint, associated with production of p210, can be seen in childhood leukemias presenting either as clinical ALL or as apparent lymphoid blast crisis of CML, suggesting that t(9;22) breakpoint locations do not exclusively determine the biologic and clinical features of pediatric Ph1-positive ALL.     

Ph1-positive childhood leukemias: spectrum of lymphoid-myeloid expressions.

Two children presented with Ph1-positive leukemia, confirmed by Giemsa banding as 22q-. One child showed an initial presentation characteristic of acute lymphoblastic leukemia, followed by development of chronic myelocytic leukemia 2 yr later. A second child presented in blast crisis. Both patients showed blast cells possessing both lymphoid and myeloid characteristics, as demonstrated by histochemical, biochemical, or surface receptor properties of each cell series. The evidence provided supports the assumption of mixed lymphoid-myeloid properties of blast cells in chronic myelocytic leukemia in children. Detailed study of the leukemic cells may aid in the understanding of complex stem cell relationships and suggest more effective therapeutic approaches.     

Molecular analysis of chromosome 22 breakpoints in adult Philadelphia-positive acute lymphoblastic leukaemia

The Philadelphia (Ph) translocation, t(9:22)(q 34:q11), is found in the majority of patients with chronic myelogenous leukaemia (CML) as well as in approximately 20% of adult acute lymphoblastic leukaemia (ALL) patients. The chromosome 22 breakpoint in CML has been localized within a restricted 5.8 kb segment of DNA known as the breakpoint cluster region (bcr). To investigate the chromosome 22 breakpoint in ALL, we analysed five adult Ph-positive ALL patients for bcr rearrangement. Rearrangement was detected within bcr in two patients. However, in one patient the break occurred 5' to the first exon of bcr and in two patients the bcr region was not involved. We conclude that the identical cytogenetic marker, t(9:22), may yield a different genomic configuration in ALL and CML.     

Clustering of breakpoints on chromosome 10 in acute T-cell leukemias with the t(10;14) chromosome translocation.

The T-cell receptor (TCR) alpha/delta chain locus on chromosome 14q11 is nonrandomly involved in translocations and inversions in human T-cell neoplasms. We have analyzed three acute T-lymphoblastic leukemia samples carrying a t(10;14)(q24;q11) chromosome translocation by means of somatic cell hybrids and molecular cloning. In all cases studied the translocation splits the TCR delta chain locus. Somatic cell hybrids containing the human 10q+ chromosome resulting from the translocation retain the human terminal deoxynucleotidyltransferase gene mapped at 10q23-q24 and the diversity and joining, D delta 2-J delta 1, regions of the TCR delta chain, but not the V alpha region (variable region of the TCR alpha chain), demonstrating that the split occurred within the V alpha-D delta 2 region. Molecular cloning of the breakpoint junctions revealed that the TCR delta chain sequences involved are made from the D delta 2 segment. The chromosome breakpoints are clustered within a region of approximately 263 base pairs of chromosome 10. The results suggest that the translocation of the TCR delta chain locus to a locus on 10q, which we have designated TCL3, results in deregulation of this putative oncogene, leading to acute T-cell leukemia.     

Clonal evolution with isodicentric Ph1 chromosome in Ph1-positive CML: Karyotypic conversion after bone marrow transplantation

Summary Clonal chromosomal evolution was observed in a 16-year-old boy suffering from Ph¹-positive CML. An isodicentric Ph¹ chromosome appeared 20 weeks after the initial diagnosis. At that time an allogeneic bone marrow transplantation was performed.Thereafter, during an observation period of more than 13 months, chromosome analyses showed neither the Ph¹ chromosome nor the abnormal isodicentric variant. Close cytogenetic monitoring is suggested to reveal early unfavourable prognostic signs of the onset of blast crisis before it becomes evident in the bone marrow morphology.     

DNA rearrangement and restriction fragment length polymorphism within the first BCR intron in Philadelphia-positive acute leukemia

In some patients with Philadelphia (Ph)-chromosome positive acute lymphoblastic leukemias, breakpoints on chromosome 22 are reported to occur within the first BCR intron. To analyze the breakpoints in chromosome 22 of Ph-positive acute leukemia patients without rearrangement of the 5.8 kb bcr, we cloned the 3' part of the first BCR intron using a synthetic DNA probe. During the course of study, we mapped the region of the deletion/insertion of 1 kb that causes a restriction fragment length polymorphism (RFLP) and found a racial difference in the frequencies of the alleles giving rise to this RFLP. Analyses of the patients' samples indicated that breakpoints were located within the 8.5 kb EcoRl fragment of the first BCR intron in two of five Ph-positive acute leukemia patients. The data, together with the previous reports, indicate that breakpoints within this approximately 50 kb intron are widely scattered, in contrast to those confined within the 5.8 kb bcr in chronic myelogenous leukemias.     

Chromosomal breakpoints within the first intron of the ABL gene are nonrandom in patients with chronic myelogenous leukemia

Bone marrow cells from 37 patients with chronic myelogenous leukemia (CML), who had the characteristic Philadelphia chromosome in their leukemic cells, were examined for ABL gene rearrangement by pulsed-field gel electrophoresis. By using several probes from the ABL gene, we found that in 33 of 37 (89%) patients studied, the translocation breakpoints in ABL fell within the 175-kilobase (kb) intron between exons 1b and 1a. Furthermore, breakpoints in this intron clustered in three regions, approximately 30 +/- 5, 100 +/- 13, and 135 +/- 8 kb downstream from exon 1b. These findings suggest that there may be specific sequences in this intron that facilitate the processes of chromosomal translocation.     

Mapping of four distinct BCR-related loci to chromosome region 22q11: order of BCR loci relative to chronic myelogenous leukemia and acute lymphoblastic leukemia breakpoints.

A probe derived from the 3' region of the BCR gene (breakpoint cluster region gene) detects four distinct loci in the human genome. One of the loci corresponds to the complete BCR gene, whereas the others contain a 3' segment of the gene. After HindIII cleavage of human DNA, these four loci are detected as 23-, 19-, 13-, and 9-kilobase-pair fragments, designated BCR4, BCR3, BCR2, and BCR1, respectively, with BCR1 deriving from the original complete BCR gene. All four BCR loci segregate 100% concordantly with human chromosome 22 in a rodent-human somatic cell hybrid panel and are located at chromosome region 22q11.2 by chromosomal in situ hybridization. The BCR2 and BCR4 loci are amplified in leukemia cell line K562 cells, indicating that they fall within the amplification unit that includes immunoglobulin lambda light chain locus (IGL) and ABL locus on the K562 Philadelphia chromosome (Ph1); additionally, in chronic myelogenous leukemia-derived mouse-human hybrids retaining a Ph1 chromosome in the absence of the 9q+ and normal chromosome 22, BCR2 and BCR4 loci are retained, whereas the 3' region of BCR1 and the BCR3 locus are lost, indicating that BCR3 is distal to BCR1 on chromosome 22. Similarly, in mouse-human hybrids retaining a Ph1 chromosome derived from an acute lymphoblastic leukemia-in the absence of the 9q+ and 22, only BCR2 and BCR4 loci are retained, indicating that the breakpoint in this acute lymphoblastic leukemia, as in chronic myelogenous leukemia, is proximal to the BCR1 3' region, but distal to the IGLC locus and the BCR2 and BCR4 3' loci. Thus, the order of loci on chromosome 22 is centromere----BCR2, BCR4, and IGL----BCR1----BCR3----SIS, possibly eliminating BCR2 and BCR4 loci as candidate targets for juxtaposition to the ABL gene in the acute lymphoblastic leukemia Ph1 chromosome.     

AICDA expression in BCR/ABL1-positive acute lymphoblastic leukaemia is associated with a peculiar gene expression profile

Activation-induced cytidine deaminase (AICDA) initiates somatic hypermutation and class-switch recombination of immunoglobulin (Ig) genes and induces mutations also in non-Ig genes. AICDA aberrant expression was detected in B-lineage acute lymphoblastic leukaemia (B-ALL), particularly BCR/ABL1+ B-ALL; patients expressing AICDA carried more copy number alterations than 'AICDA-negative' cases. To determine the role of AICDA, AICDA expression and gene expression profiling were studied in adult BCR/ABL1+ B-ALL. Patients displaying the full-length isoform AICDA are characterized by up-regulation of DNA repair/replication and cell cycle genes, suggesting their involvement in the genetic instability of BCR/ABL1+ B-ALL.     

Consistent involvement of the 3'half part of the first BCR intron in adult Philadelphia-positive leukaemia without M-bcr rearrangement

Summary About one half of Philadelphia (Ph)-positive acute leukaemia (AL) patients and rare cases of Ph-positive chronic myelogenous leukaemia (CML) show rearrangements within the first intron of the BCR gene on chromosome 22. We studied breakpoints within the first BCR intron in 22 adult patients with Ph-positive leukaemia; 21 with AL and one with CML, which lacked rearrangements within the major bcr (M-bcr). With a series of genomic probes from this intronic region, we detected chromosomal breaks in all 22 patients within the 35 kb area, corresponding almost to the 3'half portion of the intron. The breakpoints were distributed throughout this region but we could not identify any special cluster of breakpoints in this area. Our data support consistent involvement of the 3'half part of the first BCR intron in Ph-positive leukaemias without M-bcr rearrangement, and indicate relatively wide scattering of breakpoints in this portion of the intron.     

The nerve growth factor receptor gene is at human chromosome region 17q12-17q22, distal to the chromosome 17 breakpoint in acute leukemias.

Genomic and cDNA clones for the human nerve growth factor receptor have been used in conjunction with somatic cell hybrid analysis and in situ hybridization to localize the nerve growth factor receptor locus to human chromosome region 17q12-q22. Additionally, part, if not all, of the nerve growth factor receptor locus is present on the translocated portion of 17q (17q21-qter) from a poorly differentiated acute leukemia in which the chromosome 17 breakpoint was indistinguishable cytogenetically from the 17 breakpoint observed in the t(15;17)(q22;q21) translocation associated with acute promyelocytic leukemia. Thus the nerve growth factor receptor locus may be closely distal to the acute promyelocytic leukemia-associated chromosome 17 breakpoint at 17q21.     

A case of acute myeloblastic leukemia with Ph1 chromosome showing translocation 9q+;22q-.

Cytogenetic studies of the bone marrow and peripheral blood with the quinacrine fluorescence banding technique in a patient with the clinical diagnosis of acute myeloblastic leukemia revealed the Philadelphia chromosome due to a translocation between chromosomes 22 and 9. He had been exposed to the atomic bomb in Hiroshima, and some hours after the exposure he wandered into the hypocenter.     

The Philadelphia chromosome (Ph1) in adults presenting with acute leukaemia: a comparison of Ph1+ and Ph1-patients.

To evaluate the frequency and clinical significance of the Philadelphia chromosome (Ph1) in adult acute leukaemia, bone marrow chromosomes were studied in 15 adults with acute lymphocytic leukaemia (ALL) and 55 with acute nonlymphocytic leukaemia (ANLL). Morphology, clinical findings, therapeutic response and survival were compared in patients with and without the Ph1. The Ph1 was found in six newly diagnosed adults presenting with ALL. Adults with Ph1+ ALL differed from those with Ph1-ALL in being older, in having more frequent lymphadenopathy and splenomegaly and in demonstrating higher initial leucocyte counts and more peripheral blasts. Complete remissions were obtained in all nine adults with Ph1-All but in only three of six with Ph1+ ALL. Adults with Ph1-ALL survived significantly longer. Four adults with ANALL were Ph1+. They did not respond to treatment and survived significantly shorter periods than adults with Ph1-ANLL. No clinical or morphologic features indicated which patients with acute leukaemia would have the Ph1. Since the presence of the Ph1 in acute leukaemia has therapeutic and prognostic significance, marrow chromosome studies should be performed in adults presenting with acute leukaemia, especially ALL.