Analysis of breakpoints within the bcr gene and their correlation with the clinical course of Philadelphia-positive chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is characterized by a reciprocal translocation between chromosomes 9 and 22. The breakpoints on chromosome 22 are clustered within a 5.8-kilobase (kb) DNA fragment known as the breakpoint cluster region (bcr), which encodes part of a functionally active gene. We analyzed the bcr in DNAs from 108 consecutive, unselected Philadelphia chromosome-positive CML patients by Southern blot and determined five restriction enzyme fragments within which breaks occur on chromosome 22. The exact sublocalization was determined in the DNA of 100 patients. It was found to be within the 5.8-kb in 99 patients and outside the bcr in only one. Within the bcr, most of the breakpoints occurred in fragments 1, 2, and 3. Overall, laboratory and clinical features of CML did not correlate with specific breakpoint fragments, but chronic-phase duration was longer in patients with a breakpoint in fragment 2 of the bcr. Large 3' bcr deletions were found in nine patients but did not influence clinical outcome. DNA from one of six patients analyzed both during chronic phase and blastic crisis showed an additional aberrant fragment, which suggested that a second abnormal clone developed in blastic crisis.     

The site of the breakpoint within the bcr is a prognostic factor in Philadelphia-positive CML patients [see comments]

The Philadelphia (Ph1) chromosome, characteristic of chronic myelogenous leukemia (CML), arises from a reciprocal translocation between chromosomes 9 and 22. The site of the breakpoint on chromosome 22 is within a small region called the breakpoint cluster region (bcr). We have mapped the breakpoint within the bcr in peripheral blood leukocyte DNA from 22 Ph1-positive CML patients. No correlation between the site of the breakpoint and the clinical phase of the disease was found. However, a striking correlation between the site of the breakpoint and the length of time between presentation and onset of acute phase was observed: on average, patients with a 5' break-point had a fourfold longer chronic phase (median, 203 weeks) than those with a 3' breakpoint (median, 52 weeks).     

Relationship of bcr breakpoint to chronic phase duration, survival, and blast crisis lineage in chronic myelogenous leukemia patients presenting in early chronic phase

Strong evidence implicates fusion of control elements and 5' sequences of the bcr gene of chromosome 22 with 3' sequences of the c-abl gene of chromosome 9 in the pathogenesis of Ph-positive and certain cases of Ph-negative chronic myelogenous leukemia (CML). Since this fusion gene gives rise to a chimeric tyrosine protein kinase with transforming potential, and since the bcr exon contribution to this chimeric protein is variable, the question has arisen as to whether bcr breakpoint location and bcr exon contribution could influence the clinical course of CML. Prior studies have yielded conflicting results on this point. Here we have looked, in a manner approximating a prospective analysis, at the relation of bcr breakpoint localization to the duration of chronic phase, total survival, and blast crisis phenotype in 81 patients presenting in the chronic phase of CML. We have found no significant differences in chronic phase duration or total survival among patients with breakpoints in the three major subregions of a breakpoint cluster region within the bcr gene. These findings indicate that chronic phase duration and total survival cannot be predicted from bcr breakpoint for CML patients presenting in chronic phase and suggest that unknown oncogenic events determining the onset of blast crisis are the prime determinants of prognosis. Combined analysis of blast crisis cell lineage in our patients and patients presented in a previous study has revealed an overall ratio of myeloid:lymphoid (M:L) crisis of 3.4:1, but a striking predominance of myeloid crisis in patients with breakpoints in subregion 2 (M:L of 9:1), and a lower than expected M:L ratio (1.6:1) among patients with breakpoints in subregion 3 (P for subregion 2 versus 3 = .012; subregions 0,1,2 versus 3 = .012; subregions 0,1,3 versus 2 = .032). The molecular basis for this divergence from the anticipated M:L ratio in patients with breakpoints in bcr subregions 2 and 3 is unknown.     

Long-range mapping of the Philadelphia chromosome by pulsed-field gel electrophoresis

The Philadelphia chromosome (Ph1) of chronic myelogenous leukemia (CML) contains sequences from chromosome 9, including the ABL protooncogene, that have been translocated to the breakpoint cluster region (bcr) of chromosome 22, giving rise to a bcr-ABL fusion gene, whose product has been implicated in the genesis of CML. Although chromosome 22 translocation breakpoints in CML virtually always occur within the 5.8- kilobase (kb) bcr, chromosome 9 breakpoints have been identified within the known limits of ABL in only a few instances. For a better understanding of the variability of the breakpoints on chromosome 9, we studied the CML cell line BV173. Using pulsed-field gel electrophoresis (PFGE), large-scale maps of the t(9;22) junctions were constructed. The chromosome 9 breakpoint was shown to have occurred within an ABL intron, 160 kb upstream of the v-abl homologous sequences, but still 35 kb downstream of the 5'-most ABL exon. bcr-ABL and ABL-bcr fusion genes were demonstrated on the Ph1 and the 9q+ chromosomes, respectively; both of these genes are expressed. These results suggest that the 9;22 translocation breakpoints in CML consistently occur within the limits of the large ABL gene. RNA splicing, sometimes of very large regions, appears to compensate for the variability in breakpoint location. These studies show that PFGE is a powerful new tool for the analysis of chromosomal translocations in human malignancies.     

Aberrant methylation of the major breakpoint cluster region in chronic myeloid leukemia

Isolated hypomethylated sites exist in the major breakpoint cluster region (M-bcr) where most Philadelphia chromosome (Ph) breakpoints are located. Twenty of 50 (40%) chronic myeloid leukemia (CML) patients were found to have aberrant hypermethylation of these sites on the rearranged M-bcr when compared with control marrows. The aberrancy correlated strongly with M-bcr breakpoint location; 19 of 20 cases had breakpoints located 5' of the M-bcr Sca I site, and 28 of 30 cases with normal M-bcr methylation had breakpoints located 3' of the M-bcr Sca I site. Sequence analysis of the Ph M-bcr breakpoints failed to find an M- bcr nucleotide position that delineated the transition between abnormally and normally methylated cases, indicating that the translocation of a critical M-bcr sequence was not responsible for the methylation abnormality. In 3 of 8 CML patients, cells without the t(9;22) were found to have abnormally methylated, unrearranged M-bcrs. The data indicate that abnormally methylated rearranged M-bcrs are present in CML cases with Ph breakpoints 5' of the M-bcr Sca I site and that the M-bcr in Ph- cells of patients with CML may also be abnormally methylated.     

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.     

Micro-osteoclast resorption as a characteristic feature of B-cell malignancies other than multiple myeloma

Among 77 unselected patients with chronic myelogenous leukaemia (CML), 70 had Philadelphia chromosome (Ph1) in blood cells. Extra chromosomal abnormalities were noted in 4%, 55% and 78% of Ph1-positive patients in chronic phase, accelerated phase and acute blast crisis, respectively. Rearrangement of the bcr was detected in 46 of 47 Ph1-positive patients studied and also in three of five Ph1-negative ones. The locations of the breakpoints were mapped to one of four zones of the bcr in 45 patients. The median duration from diagnosis of CML to onset of acute blast crisis was not significantly different between the two groups of patients with breakpoints in the 5' portion (34 months), and in the 3' portion (39 months) of the bcr. In addition, the locations of the breakpoints within the bcr did not change as the disease progressed in the six patients who had DNA analysed both in the chronic phase and subsequently in transformation. In one of them, an additional aberrant band which was not present in the beginning of the acute phase was detected in blood cells taken 2 months later. It is suggested from the studies that transformation of CML may not be related to alterations within the bcr.     

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.     

Ph1-positive, bcr-negative acute leukemias: clustering of breakpoints on chromosome 22 in the 3' end of the BCR gene first intron

About 50% of the Philadelphia-positive acute leukemias undergo molecular rearrangements outside the now classical bcr sequence (or M-BCR-1) rearranged in chronic myeloid leukemia (CML). Most of the breakpoints on chromosome 22 have been shown to be clustered in a 10.8-kb region of the first intron of the BCR gene (called bcr2 or m-BCR-1). In this report we examined two cases of Ph1 acute lymphoblastic leukemia in adult patients that exhibited breakpoints in a 5-kb segment of the BCR gene first intron, 16 kb upstream of the previously described cluster, suggesting the possibility of a second minor breakpoint cluster. In addition, the breakpoints on chromosome 9 were located in a region just 5' of the c-abl exon la.     

DETECTION OF REARRANGEMENT WITHIN THE BREAKPOINT CLUSTER REGION OF CHROMOSOME 22 IN THE DIAGNOSIS OF CHRONIC MYELOID LEUKEMIA

Chronic myeloid leukemia (CML) is characterised by the presence of a Philadelphia (Ph) chromosome in approximately 95% of patients. Molecular analysis has shown that the Ph chromosome translocation breakpoints are clustered within 5.8kb on chromosome 22 (breakpoint cluster region or bcr). This has facilitated the diagnosis of CML by nucleic acid hybridisation using probes specific for the bcr to detect DNA rearrangement in this region. Forty patients diagnosed with CML, including four with variant Ph chromosome translocations and three with normal karyotypes were analysed for rearrangement within the bcr. All except one patient with Ph negative CML had rearrangement within the bcr. In contrast, none of the patients diagnosed with other hematological disorders such as the myelodysplastic or myeloproliferative syndromes (16 patients), acute myeloid leukemia (AML) (six patients), acute lymphoblastic leukemia (ALL) (five patients), including Ph positive ALL (two patients), showed rearrangement within the bcr. Analysis for rearrangement within the bcr is useful in the diagnosis of CML, especially when cytogenetic analysis is unsuccessful or in patients with normal karyotypes or variant Ph chromosome translocations.     

Identification of molecular variants of p210bcr-abl in chronic myelogenous leukemia

The aberrant abl protein product of a chronic myelogenous leukemia (CML) blast crisis cell line (K562) and of five Philadelphia chromosome- positive CML patients in blast crisis were analyzed by an immune complex kinase assay using two antipeptide sera generated against the hydrophilic domain of v-abl and a region within the third exon of the breakpoint cluster region (bcr) respectively. Both the anti-abl and anti-bcr sera detected a 210 kd band in extracts derived from K562 cells and from two CML patients with myeloid blast crisis. p210 was detected by the anti-abl but not the anti-bcr sera in three CML patients with myeloid (one patient) and lymphoid (two patients) blast crisis, indicating the absence of bcr exon 3 in this protein. Southern blot analysis on DNA derived from one of the patients in the latter group was consistent with the break on chromosome 22 occurring 5' to bcr exon 3. Our observations demonstrate that the Philadelphia translocation results in the generation of a chimeric bcr-abl protein with at least two molecular variants, both of which are enzymatically active as protein kinases.     

BCR rearrangement and cytogenetic findings in Philadelphia-positive chronic myelocytic leukemia in children

Eight children with Philadelphia (Ph1) chromosome positive chronic myelocytic leukemia (CML) were available for cytogenetic studies and breakpoint cluster region (bcr) rearrangement analysis as compared to the features of adults with Ph1-positive CML. In chronic phase additional abnormalities other than Ph1 chromosome were found in none of our cases. On the other hand, in blastic crisis all of 6 cases had additional chromosomal abnormalities like as i(17q), double Ph1 and +8. The frequency of the appearance of additional chromosomal abnormalities, especially i(17q), is higher in children than in adult cases. In the DNA of 7 of 7 examined patients, rearrangement of bcr could be demonstrated by Southern blot analysis. These findings were similar to those observed in adults. An analysis of the location of the bcr breakpoint indicated that 5' breakpoints were found in four cases who were long-term survivors, and two of the other cases had blastic crisis from the onset of the disease. These findings showed the cytogenetic findings of children with Ph1+CML were different from those of the adult cases in the frequency of the appearance of the additional chromosomal abnormalities, and the location of the bcr breakpoint in children cases might be different from that in the adult cases and influence its prognosis.     

Essential thrombocythaemia and the Philadelphia chromosome

Six adult patients presented with clinical features of essential thrombocythaemia. Five of the patients, although Ph-positive, have maintained these features without evidence of leukaemia; in one case for 9 years. A sixth patient developed leukaemic blast crisis following a persistently high platelet count over 4 years. Her cells were Ph-negative, but hybridization of gene probes to chromosomes in situ and to leukaemic DNA showed that the abl oncogene had moved to the breakpoint cluster region (bcr) on the normal chromosome 22. This patient has the same molecular gene change as occurs in some cases of Ph-negative chronic myeloid leukaemia (CML) whose leukaemic cells likewise show no evidence of chromosomal translocation. Molecular studies are essential for the correct diagnosis of these patients. The Ph genomic lesion appears to have a range of leukaemic expression which includes thrombocythaemia as well as chronic myeloid leukaemia and acute lymphatic leukaemia.     

Localization of the human c-sis oncogene in Ph1-positive and Ph1-negative chronic myelocytic leukemia by in situ hybridization

Oncogenes are a group of evolutionary conserved cellular genes (c-onc) homologous to the transforming genes of oncogenic retroviruses (v-onc). Some of them are localized near the breakpoints of specific chromosomal aberrations occurring in various neoplasms, as for example the Philadelphia translocation, t(9;22)(q34;q11), in chronic myelocytic leukemia (CML). Recently, we localized the human c-abl oncogene to chromosome region 9q34 and demonstrated a translocation of this gene to the Philadelphia chromosome (Ph1,22q-) in various forms of Ph1-positive, but not Ph1-negative, chronic myelocytic leukemia (CML). Another human oncogene, c-sis, is located on chromosome 22 and was recently reported to be transferred to chromosome 9q+ in one CML patient. We have now studied 2 CML patients with classic and variant types of Ph1 translocation, one Ph1-negative case, and a healthy control using in situ hybridization of a c-sis probe to metaphase chromosomes. These studies show that c-sis: (1) is localized to region 22q12.3-q13.1, far away from the breakpoint region 22q11 in CML, (2) segregates with the translocated part of chromosome 22 to different chromosomes in Ph1-positive patients, and (3) remains on chromosome 22 in the Ph1-negative case. Therefore, these data give no support for an active role of the c-sis gene in the generation of CML. Thus, if either of these two oncogenes is involved in the development of Ph1-positive CML, c-abl appears to be the more important one.     

The bcr gene in Philadelphia chromosome positive acute lymphoblastic leukemia

In chronic myelogenous leukemia (CML) and in a percentage of childhood and adult acute lymphoblastic leukemia (ALL) the Philadelphia (Ph') chromosome is present in the leukemic cells of patients. This chromosome is the result of a reciprocal translocation between chromosomes 9 and 22. In CML the break on chromosome 22 occurs within the major breakpoint cluster region (Mbcr) of the bcr gene. In this study, we report on the examination of DNAs from nine Ph'-chromosome positive ALL patients for rearrangements within the bcr gene using Southern blot analysis. Of nine patients having a karyotypically identifiable Ph'-chromosome, only five exhibited rearrangements of the bcr gene. This could indicate that in ALL, chromosome 22 sequences other than the bcr gene are involved in the Ph'-translocation. Within the group of Ph'-positive ALL patients having a bcr gene breakpoint, a correlation appears to exist between the age of the patient and the location of the breakpoint within the gene: all or the vast majority of pediatric patients analyzed to date do not have a Mbcr breakpoint as found in CML and in adult ALL.     

Acquired homozygosity of the rearranged bcr allele during the acute leukemic phase of a patient with Ph-negative chronic myeloid leukemia

A 45-year-old male patient with Ph-negative chronic myeloid leukemia (CML) had rearranged bcr-3' and bcr-5' gene regions in Southern blot studies when leukemia was diagnosed. During development of terminal blast crisis, successive blood samples showed a progressive decrease in the amount of germline bcr DNA and its complete loss by full blast crisis. There were also increased amounts of rearranged bcr DNA consistent with acquired homozygosity. A similar result was obtained with an IgV lambda probe and indicated homozygosity of a significant part of chromosome 22. The bcr-abl gene complex behaves as a somatic dominant in CML, and we suggest that its acquired homozygosity is a mechanism of bcr-abl amplification similar to duplication of the Ph chromosome commonly found in the blast crisis of CML.     

Application of a bcr-specific probe in the classification of human leukaemia

Summary A genomic probe derived from the breakpoint cluster region (bcr) on chromosome 22q11 was used to assess whether Philadelphia (Ph) chromosome positive chronic myelogenous leukaemia patients have unique patterns of bcr rearrangements and whether this pattern is modified as the disease progresses from stable phase to blast crisis. The data indicated that bcr rearrangements are fairly unique to each patient and are not subject to additional modifications during the course of the disease. We have also found bcr rearrangements in acute lymphocytic leukaemia (ALL) patients, usually of the cALL phenotype. For the majority of Ph⁺ ALL patients, the breakpoint on 22q11 was in bcr. However, we describe a case of Ph⁺ ALL without bcr rearrangement, indicating heterogeneity of Ph chromosomes in ALL at the molecular level. Contrary to previous reports, a bcr rearrangement was also identified in a childhood cALL.