The BCR–ABL fusion in chronic myeloid leukemia (CML) is represented in approximately 95% of all CML by the b2a2- and the b3a2-fusion transcripts (M–BCR), whereas the b2a3-, b3a3-, e1a2-, e19a2- and other rare fusions each account for less than 1%.1 One of the rare fusion transcript types is the e6a2 with breakpoint in intron 6 of BCR. So far, this fusion type was reported in 10 CML cases and in one T-acute lymphoblastic leukemia (T-ALL) case only.2, 3, 4 Here we contribute another case of e6a2 in chronic phase of CML which developed resistance to imatinib and subsequent dasatinib treatment.
In July 2006, a 48-year-old male patient presented with increased left-shifted granulopoiesis and basophils suspicious of CML (leukocytes 31 × 109 l−1; hemoglobin 12.5 g dl−1; platelets 260 × 109 l−1). Bone marrow morphology showed CML in chronic phase. Interphase fluorescence in situ hybridization (FISH) confirmed the BCR–ABL fusion in 90/100 nuclei. In chromosome banding analysis in 12 of 25 metaphases, one chromosome 22 showed a deletion of the long arm while both chromosomes 9 appeared normal. Additional FISH with probes for chromosome 1, 9 and 22 as well as BCR and ABL hybridized on metaphases revealed an insertion of material of the long arm of chromosome 22 into the short arm of chromosome 1 and into the long arm of chromosome 9 with a colocalization of BCR and ABL on the long arm of one chromosome 9. Therefore, the karyotype is described as 46,XY,del(22)(q11q13).ish,der(1)ins(1;22)(p2?2;q11q13)(BCR+),der(9)ins(9;22)(q34;q11q11)(ABL+,BCR+),del(22)(q11q13)(BCR−) (Figure 1). Standard multiplex reverse transcription polymerase chain reaction (RT-PCR) failed to detect the BCR–ABL fusion. With alternative primers5 a 1000 bp product could be amplified. Direct sequencing of this PCR product identified the rare e6a2 transcript variant. Imatinib was started at a dosage of 400 mg. Three months later, interphase FISH still revealed BCR–ABL in 59/100 nuclei in peripheral blood. As quantitative RT-PCR optimized for e6a2 was not available, we performed an assay optimized for the e1a2-fusion. This assay had a relatively low sensitivity of 1:100 for e6a2 but was able to show a slight decrease of BCR–ABL/ABL transcripts (from 0.224 to 0.063). Six months after the start of imatinib, cytogenetics showed the aberrant clone with an additional tetrasomy 8 and trisomy 15 (49,XY,+8,+8,+15,del(22)(q11q13)) implicating clonal evolution in the Philadelphia-positive clone in all 13 metaphases (interphase FISH: 87/100 BCR–ABL+ nuclei; BCR–ABL/ABL ratio: 0.225). Sequencing of the ABL part of the BCR–ABL fusion transcript revealed two novel BCR–ABL kinase domain mutations: a K245E in 25% and an L284S mutation in 50% of all fusion transcripts, both in the P-loop of the tyrosine kinase (Figure 2). At this time the patient showed increasing WBCs and developed fever above 39 °C without the evidence of infection. Hydroxyurea was given to control the leukocytosis but the patient developed severe mucosal ulcerations leading to perforation of the colon and requiring emergency surgery. Due to this fact therapy was changed to dasatinib (2 × 70 mg). This treatment led to normalization of peripheral blood count and complete disappearance of fever. Follow-up in March 2007 revealed complete hematological and major cytogenetic remission (3/20 Philadelphia Chromosome positive (Ph+) metaphases; interphase FISH: 11/100 BCR–ABL+ nuclei).
However, after 5 months of dasatinib, peripheral blood again showed an increase of leukocytes (15.1 × 109 l−1 and loss of hematological remission. Cytogenetics revealed 18/20 Ph+ bone marrow metaphases (interphase FISH: 60/100 BCR–ABL+ nuclei; BCR–ABL/ABL 0.325 similar to the diagnostic value). Mutation analysis showed reduction of the K245E and L284S mutated clones below the detection level. Instead, a T315I mutation was detected in almost 100% of all fusion transcripts. This mutation is known to confer resistance to imatinib, dasatinib and nilotinib. The patient rapidly progressed to blast crisis and died. The originally planned allogeneic stem cell transplantation could not be performed (Table 1).
The comparison of our patient with other reported cases leads to some important conclusions: including this case 10/11 reported cases were men.6, 7 The complex variant Philadelphia translocations in this and in the T-ALL case4 show that Philadelphia translocations might be as diverse in cases with e6a2 as in those with the frequent transcript types. So far, cryptic BCR–ABL rearrangements,1, 4 additional chromosomal aberrations4, 8 or variant translocations were reported in three cases with e6a2, whereas 7 of 10 previously reported cases revealed standard Philadelphia translocations. As 5 of 10 previously reported patients with e6a2 had de novo blast crisis or chloromes,1, 6, 8, 9 a higher frequency of advanced phases and a worse prognosis were suggested.2 It was hypothesized that the localization of the BCR breakpoint probably in the middle of the GEF/dbl-like domain might mediate interaction with cell growth and signaling enhancing the oncogenic potential of the BCR–ABL protein.9 Others suggested that prognosis might be less adverse with imatinib,7 as 6 of 8 CML patients (including this case) showed good initial response.6, 7, 8, 9 Our patient showed two different imatinib resistance mediating mutations in the loop of the kinase (K245E and L284S), which were not described so far and which were replaced by a T315I under dasatinib treatment. Whether different transcript types are associated with distinct imatinib resistance mutations remains open. The combination of the variant translocation and Philadelphia-positive clonal evolution, and the diverse resistance mutations in this case suggests that therapy failure was caused by several mechanisms. The submicroscopic 9q-deletion might have played an additional role, as breakpoint-spanning deletions might worsen prognosis in CML.10
This case is a further example that shows the necessity to combine cytogenetics, interphase FISH, and optimized RT-PCR to get a comprehensive genetic evaluation of a CML under modern treatment options.
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Schnittger, S., Bacher, U., Kern, W. et al. A new case with rare e6a2 BCR–ABL fusion transcript developing two new resistance mutations during imatinib mesylate, which were replaced by T315I after subsequent dasatinib treatment. Leukemia 22, 856–858 (2008). https://doi.org/10.1038/sj.leu.2404949
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