Heterogeneity of genomic fusion of BCR and ABL in Philadelphia chromosome-positive acute lymphoblastic leukemia.

Abstract

Philadelphia chromosome-positive acute lymphoblastic leukemia occurs in two molecular forms, those with and those without rearrangement of the breakpoint cluster region on chromosome 22. The molecular abnormality in the former group is similar to that found in chronic myelogenous leukemia. To characterize the abnormality in the breakpoint cluster region-unrearranged form, we have mapped a 9;22 translocation from the Philadelphia chromosome-positive acute lymphoblastic leukemia cell line SUP-B13 by using pulsed-field gel electrophoresis and have cloned the DNA at the translocation junctions. We demonstrate a BCR-ABL fusion gene on the Philadelphia chromosome. The breakpoint on chromosome 9 is within ABL between exons Ia and II, and the breakpoint on chromosome 22 is :50 kilobases upstream of a breakpoint cluster region in an intron of the BCR gene. This upstream BCR breakpoint leads to inclusion of fewer BCR sequences in the fusion gene, compared with the BCR-ABL fusion gene of chronic myelogenous leukemia. Consequently, the associated mRNA and protein are smaller. The exons from ABL are the same. Analysis of leukemic cells from four other patients with breakpoint cluster regionunrearranged Philadelphia chromosome-positive acute lymphoblastic leukemia revealed a rearrangement on chromosome 22 close to the breakpoint in SUP-B13 in only one patient. These data indicate that breakpoints do not cluster tightly in this region but are scattered, possibly in a large intron. Given the large size of BCR and the heterogeneity in breakpoint location, detection of BCR rearrangement by standard Southern blot analysis is difficult. Pulsed-field gel electrophoresis should allow detection at the DNA level in every patient and thus will permit clinical correlation of the breakpoint location with prognosis.A Philadelphia chromosome (Ph') resulting from a 9;22 translocation, t(9;22)(q34;qll), characterizes the malignant cells in virtually all patients with chronic myelogenous leukemia (CML) (1, 2), in 10% of those with acute lymphoblastic leukemia (ALL) (3), and in rare cases of acute myelogenous leukemia (4). The t(9;22) is the most common chromosomal abnormality in adults with ALL (3); it is found in one of five of these patients and in well over half of those with specific chromosomal translocations. In children with ALL, -6% are found to have the t(9;22). In CML and in a subgroup of Ph'-positive ALL, the breakpoints of the translocation occur in a 200-kilobase (kb) area within the 5' introns of the ABL gene (5) and in the 5.8-kb breakpoint cluster region (bcr) of the BCR gene (6, 7) on chromosomes 9 and 22, respectively. This results in a 5' BCR-ABL 3' fusion gene on the Ph' (8,9) that is expressed as an 8.5-kb hybrid BCR-ABL mRNA. The translation product of the hybrid message is a 210-kDa tyrosine kinase (P210BCRABL) (10). The P210 is thought to play a central role in the malignant process (11). In contrast, in many cases of Ph'-positive ALL, the breakpoint on chromosome 22 is not in a bcr (12)(13)(14), and the 8.5-kb ...

Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi)

“…The translocation fuses two unrelated genes, BCR from chromosome 22 and ABL from chromosome 9, to form an oncogenic hybrid gene. With regard to the BCR gene, there are two breakpoint sites, M-BCR versus m-BCR, and hence two slightly different BCR/ ABL oncoproteins of 210 kD or 190 kD, respectively, are produced (Chan et al, 1987;Rubin et al, 1988;Hooberman et al, 1989). The resulting BCR/ABL protein has, as compared to the normal ABL protein, an increased kinase activity leading to pathological phosphorylation of several downstream targets (Lugo et al, 1990;Goldman and Druker, 2001a).…”

Unexpected heterogeneity of BCR-ABL fusion mRNA detected by polymerase chain reaction in Philadelphia chromosome-positive acute lymphoblastic leukemia.

“…Gels were stained with ethidium bromide, photographed, and transferred to GeneScreenPlus nylon membranes. Blots were hybridized in a solution containing 15-30o formamide, 1 M NaCl, 50 mM sodium phosphate (pH 6.5), 1% NaDodSO4, 5X Denhardt's solution (1x Denhardt's solution = 0.02% bovine serum albumin/0.02% Ficoll/0.02% polyvinylpyrrolidone), sonicated salmon sperm DNA (200 ,tg/ml), and oligonucleotide probes (2-4 x 106 dpm/ml) specific for the amplified junctions (11,12,16) (17) and are the subject of a more detailed analysis presented elsewhere (18 and all were shown to have genomic rearrangements within bcr by Southern blot analysis.…”

“…The diagnosis of blast phase CML depended on the presence of a preexisting chronic phase. Controls used were the CML cell lines BV173 and K562, which have the bcr exon 2 to ABL exon II (bcr2-ABL) and bcr exon 3 to ABL exon II (bcr3-ABL) rearrangements, respectively (13,28), and the ALL cell line SUP-B13, which has a BCR exon 1 to ABL exon II (BCRI-ABL) rearrangement (17).…”

“…In the remaining "bcr-negative" cases, chromosome 22 breakpoints occur at least 50 kb 5' of the bcr, within the large first intron of the BCR gene ( Fig. 1 Upper) (16)(17)(18). In these instances, the resulting BCR-ABL fusion gene is transcribed into a 7-kb chimeric message consisting of only the most 5' exon of the BCR gene fused to ABL sequences beginning with exon II, which is translated into a 190-kDa BCR-ABL protein (16,(19)(20)(21)(22)(23)(24).…”

Localization of preferential sites of rearrangement within the BCR gene in Philadelphia chromosome-positive acute lymphoblastic leukemia.

“…This results in the formation of both hybrid BCR/ABL transcripts and proteins (11,12). In CML, this rearrangement has been mapped to within a 5-to 6-kilobase pair (kb) BCR (23,24). Breakpoint locations of the majority of Ph' ALL have eluded molecular definition.…”