Philadelphia-positive (Ph+) hematological malignancies are characterized by different BCR-ABL rearrangements. Three breakpoint cluster regions on chromosome 22 have been described: major (M-BCR), involving BCR exons 13 or 14 (e13 and e14, or b2 and b3) and giving origin to protein P210; minor (m-BCR), if BCR exon 1 (e1) is fused with ABL and resulting in hybrid protein P190; and micro (μ-BCR), involving BCR exon 19 (e19) and producing the rare P230. On chromosome 9 the breakpoint portion is generally positioned upstream from ABL exon 2 (a2) and only rarely upstream from exon 3 (a3).
The vast majority of chronic myeloid leukemia (CML) patients express either b2/a2 or b3/a2 transcripts; only 4 patients with M-BCR lacking ABL exon 2 (ie, b2/a3 or b3/a3) have been described.1-4 Five more cases of pediatric acute lymphoblastic leukemia with similar or m-BCR (e1/a3) hybrid gene have also been reported.2,5-7
We report the cases of 2 Ph+ CML patients who express the b3/a3 fusion transcript.
The first patient is a 69-year-old male who was diagnosed as having chronic-phase CML in April 1996: Hb 13.3 g/dL, platelet count 527 × 109/L, WBC count 18.0 × 109/L (82% neutrophils, 8% eosinophils, 4% basophils, 6% lymphocytes); bone marrow biopsy showed trilinear hyperplasia with eosinophilia. Serum chemistry was normal; epatosplenomegaly was absent. The karyotype analysis showed a three-way Philadelphia translocation 46, XY, t(4;9;22)(q31;q34;q11) in 10 mitoses, and normal pattern in 2 mitoses. The patient was initially observed for 9 months, after which, due to progressive leucocytosis and thrombocytosis, hydroxyurea therapy was started (initial dose 1 g/day, then 1.5 g/day); hematological remission was obtained, which lasted for 28 months. In May 1999 WBC and platelet counts rised again, and bone marrow biopsy was substantially stable; hydroxyurea was replaced by busulfan and 6-mercapthopurine. The patient is currently alive and well, in first chronic phase.
Reverse transcriptase polymerase chain reaction (RT-PCR) analysis using BCR exon 13 (b2) and ABL exon 3 (a3) primers was performed at diagnosis: the result was a fragment 174 bases shorter than b2/a2 and 100 bases shorter than b3/a2. This suggested a possible deletion of ABL exon 2, resulting in a b3/a3 junction, which was confirmed sequencing the target band.
The second patient is a 51-year-old male with a diagnosis of chronic-phase CML in July 1989: Hb 15.1 g/dL, platelet count 566 × 109/L, WBC count 19.9 × 109/L (circulating metamyelocytes); karyotype analysis showed a classic Philadelphia translocation 46, XY, t(9;22)(q34;q11), whereas molecular studies were not performed. After 3 months interferon (IFN) therapy was started, obtaining in 11 months a major cytogenetic response (70% Ph− metaphases). IFN was withdrawn, and autologous bone marrow transplantation (ABMT) was performed. Two months later karyotype analysis showed 70% Ph+ cells, and IFN was resumed at 9 MU/day; 16 months later (April 1992) major cytogenetic response (73% Ph−) was achieved. The status was maintained (last karyotype analysis performed in January 2000), with a complete cytogenetic response (0% Ph+ metaphases) since January 1997. The daily IFN dose was progressively reduced, down to the current dose of 3 MU/week.
Evaluation of BCR-ABL transcript was firstly performed in June 1996 (83 months after diagnosis), in major cytogenetic response: following the same technique described above, presence of a b3/a3 junction was revealed. Three more samples, collected between January 1997 (first evidence of complete cytogenetic response) and June 1999, were negative for BCR-ABL rearrangement, both at first and second step of the “nested” RT-PCR. The last sample (January 2000) is weakly positive, indicating the recurrence of the same b3/a3 transcript.
ABL exon 2 encodes for part of the SH3 domain, which is generally considered to play a negative regulatory role for the tyrosine kinase activity (SH1 region). But recent data suggest that, although “in vitro” SH3 is not required in the BCR-ABL protein for growth factor–independent proliferation, inhibition of differentiation, and protection from apoptosis, “in vivo” lack of SH3 reduces the leukemogenic potential. In addition, proliferation rate and binding capability to extracellular matrices is lower in SH3-deleted BCR-ABL expressing hematopoietic cells than in those with wild-type BCR-ABL.8
The long-lasting and quite indolent disease history of these 2 patients, supported by the evidence of a complete cytogenetic and molecular response obtained with IFN therapy in patient 2, are in agreement with the data of this study and do not confirm the hypothesis of a possible correlation between the lack of ABL exon 2 in BCR-ABL rearrangement and a more aggressive form of CML, as described by others.2,3,5-7