The vast majority of patients with chronic myelogeneous leukemia (CML) (>95%) has a breakpoint in the M-bcr, resulting in b3-a2, b2-a3, b2-a2 or b3-a3 fusion gene transcripts and the associated p210 BCR-ABL protein.1 In rare cases of CML, the breakpoint in BCR is located in the m-bcr, resulting in an e1-a2 or e1-a3 fusion gene transcript and associated p190 protein or in the μ-bcr region, resulting in an e19-a2 fusion gene transcript and the associated p230 protein. Some unusual breakpoints have been described in CML as well, including cases with transcripts derived from fusion genes in which the breaks are located within exons and cases with transcripts in which intronic sequences are inserted.1 Here, we present a 16-year old girl with CML who showed a new type of fusion gene transcript, containing part of BCR exon 18 linked to ABL exon 2, with insertion of 10 nucleotides from intron 1b at the joining site.
A 16-year old girl was admitted to the hospital because of fatigue, headache and visual abnormalities. Liver and spleen enlargement were found at physical examination. Laboratory investigation showed a hemoglobin of 4 mmol/l, a white blood cell count (WBC) of 399 × 109/l and a platelet count of 698 × 109/l. Morphologic evaluation of peripheral blood showed 4% basophils, 3% eosinophils, <1% monocytes, 2% lymphocytes, 5% blasts and 86% myeloid cells at different stages of maturation. The Sokal score was 1.57 (high risk) and the Euro score was 1459 (intermediate risk). Karyotyping showed 46,XX,t(9;22)(q34;q11) in all metaphases. The presence of a BCR-ABL fusion gene was confirmed with FISH (BCR/ABL ES probe set, Vysis). In addition, an accompanying deletion of a part of ABL was detected in the der(9)t(9;22) by this assay. Based on these findings, a diagnosis of CML in chronic phase was established.
Treatment was started with leucapheresis and hydroxyurea. After 2 weeks, the WBC had dropped to normal values and treatment was switched to imatinib 600 mg/day, according to the Dutch Childhood Oncology Group CML protocol (imatinib dose: 400 mg/m2/day). Because of thrombocytopenia, imatinib was dose-reduced to 400 mg/day at day +37. At day +56 from diagnosis the patient was in complete hematological remission. Cytogenetic analysis of the bone marrow showed 41% t(9;22)-positive metaphases. FISH analysis showed the BCR-ABL fusion in 29% of interphase cells. A major cytogenetic response was established at day +106 with 6% t(9;22) positive metaphases. At that time, a real-time quantitative (RQ)—polymerase chain reaction (PCR) analysis using the primer-probe set developed within the Europe Against Cancer program2 was performed on the diagnostic specimen as well as the bone marrow sample taken at day +106. This RQ-PCR showed an unexpectedly low amplification in the diagnostic sample and could not detect BCR-ABL fusion gene transcripts in the day +106 bone marrow. This prompted us to analyze the BCR-ABL fusion gene transcript in more detail. Therefore, PCR analysis was performed using the BIOMED-1 primer set (primer BCR-b1-A and ABL-a3-B).3 This resulted in a PCR product of approximately 800 bp, in contrast to the p210 positive control (BV173) showing the expected PCR product of 400 bp. The PCR product of the patient was cut out of the gel, sequenced, and the obtained sequence was aligned with the germline BCR and ABL sequences (accession numbers: BCR: U07000; ABL: U07563). This showed that the fusion gene transcript in the patient contained part of exon 18 of the BCR gene (the first 90 nucleotides, the last 20 nucleotides being deleted), an insertion of 10 nucleotides, followed by exon 2 of the ABL gene (Figure 1). The breakpoint at the DNA level was analyzed by ligation-mediated PCR.4 This showed that exon 18 of BCR was coupled to the first 423 bp of intron 1b downstream of ABL exon 2, resulting in a putative splice site (Figure 1).
The new in-frame e18-a2 BCR-ABL transcript is 390 base pairs larger than the e14-a2 transcript (M-Bcr) and is predicted to encode a protein 130 amino acids longer than the p210 protein. Western blot analysis, using an ABL antibody (8E9), indeed showed the presence of a protein with a molecular mass of approximately 225 kDa (data not shown). Wild-type BCR-ABL protein (p210) was not found, which is in line with the lack of a normal BCR-ABL fusion transcript. Using a flow cytometric immunobead assay (Weerkamp et al., Haematologica 2006; 91(s1):377, abstract), BCR-ABL protein was detected as well (data not shown). It should be noted that the predicted e18-a2 BCR-ABL protein contains exactly the same ABL domains as the p210 (and p190) protein, suggesting that imatinib binding will not be affected.
Based on the identified fusion gene transcript, an exon 18 specific forward primer was designed (5′-IndexTermAGGATGCCGTCCCGAAA-3′). In combination with the EAC reverse primer and probe, this resulted in a good amplification of the e18-intron 1b-a2 fusion gene transcript. MRD analysis of the day +106 bone marrow sample using this newly designed RQ-PCR showed a tumor load of 2 × 10−2 (Table 1). MRD analysis of a bone marrow sample obtained at day +210 showed a further reduction of the tumor load to 2 × 10−3. At day +239, the imatinib dose was escalated to 600 mg without recurrence of hematological toxicity. MRD analysis at day +285, day +377, and day +474 showed that the tumor load in the bone marrow remained relatively stable around a level of approximately 1 × 10−3 (Table 1). Imatinib therapy will be continued and regular follow-up for the occurrence of resistance is planned.
To our knowledge, only seven CML patients in whom transcripts are derived from fusion genes in which the breaks are located within exons have been described so far. In two of these patients (a 65-year-old male and a 59-year-old male), the unusual transcript consisted of 86 base pairs of BCR exon 15 joined in frame with ABL exon 2 at nucleotide 78.5 These transcripts could only be identified upon nested PCR approaches and it was hypothesized that they only occurred in a small subset of cells. Both patients showed mild clinical symptoms (moderate leukocytosis that did not increase with time) and an indolent clinical course. A third patient (a 49-year-old male) showed BCR-ABL transcripts in which part of BCR exon e2 was joined to part of ABL exon 1a; this patient had an aggressive clinical course.6 A fourth patient (a 51-year-old male) showed an in-frame fusion consisting of part of BCR exon e8 spliced to ABL exon a2.7 Finally, one patient (a 31-year-old male) was reported to have a break in BCR exon 3 and two patients (45-year-old and 56-year-old women) were reported with a break in BCR exon 8, in all three cases linked to ABL exon 2.7, 8 Of interest, the latter three patients had an inserted sequence (15-44 nucleotides) derived from intron 1b in between BCR and ABL.7, 8
The here presented case is of interest for various reasons. It is the first patient with a breakpoint within BCR exon 18. Secondly, in contrast to most other reported patients with breaks within exons, the BCR-ABL transcript in our patient contained an insertion of 10 nucleotides of intron 1b between BCR exon 18 and ABL exon 2. Third, it is remarkable that in contrast to the seven previously described patients, all adults, our case concerns a 16-year-old female. Finally, this case demonstrates that also a patient with the non-classical e18-a2 BCR-ABL fusion can respond to imatinib.
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We thank Marieke Comans-Bitter for preparation of the figure and dr. Floor Weerkamp and Karin Brouwer-de Cock for performing the protein analysis.
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van der Velden, V., Beverloo, H., Hoogeveen, P. et al. A novel BCR-ABL fusion transcript (e18a2) in a child with chronic myeloid leukemia. Leukemia 21, 833–835 (2007). https://doi.org/10.1038/sj.leu.2404580
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