TO THE EDITOR
Chronic myelomonocytic leukemia (CMML) is characterised by a persistent peripheral blood monocytosis (above 1 × 109/l) and the absence of a Philadelphia (Ph) chromosome (and BCR-ABL fusion gene). Molecular analysis of the Ph chromosome, identified in more than 95% of patients with chronic myeloid leukemia (CML), has shown that, according to the breakpoints in chromosome 22, the translocation t(9;22)(q34;q11) gives rise to a diversity of BCR-ABL fusion proteins involved in the pathogenesis of the disease (Figure 1a). Most CML patients express the BCR-ABL transcript with the junctions b3a2 (e14a2) or b2a2 (e13a2) corresponding to the major BCR gene breakpoint cluster region (M-bcr). A few CML patients express a shortened BCR-ABL transcript with an e1a2 junction (m-bcr) and only sporadic cases express the rare junctions b2a3, b3a31 or e19a2 (μ-bcr). Interestingly, most of these rare CML cases are associated with a particular clinical phenotype (a significant monocytosis for e1a2 junction2, 3 and a clinical picture of neutrophilic-CML for the e19a2 junction4). These various CML clinical phenotypes with an e1a2 and e19a2 junctions raise the question of whether different forms of the BCR-ABL protein (heterogeneous by their BCR domain contribution) have intrinsically different leukemogenic activities in hematopoietic cells and may influence the clinical features of the disease entity (Figure 1a). Another atypical BCR-ABL transcript with an e6a2 junction (between e1a2 and b2a2 junctions) has only been described in four CML patients to date.5, 6, 7, 8 None of these cases showed associated monocytosis or additional chromosomal abnormalities. The clinical and hematologic features of these previously reported cases have been recently reviewed and according to the authors could be associated with a worse prognosis.8 In this paper, we report for the first time to our knowledge, a CMML patient, Philadelphia negative at diagnosis, who acquired this translocation along the course of the disease. Molecular studies allowed us to identify and quantify a rare transcript, the e6a2 BCR-ABL fusion mRNA in this CMML case.
A 64-year-old female patient was referred to our department in July 2001 for a thrombocytosis at 622 × 109/l and a monocytosis at 1.9 × 109/l discovered on a systematic peripheral blood analysis. Other leukocyte subsets counts and hemoglobin level were normal. Hepatosplenomegaly and lymphadenopathy were absent. At that time, the bone marrow morphology was considered as normal. A second bone marrow aspirate performed on March 2002 displaying myelodysplastic features associated with 15% of monocytes, consistent with the diagnosis of CMML. Standard cytogenetics by Giemsa banding showed a normal karyotype (46,XX) in two bone marrow samples performed during the first 2 years following diagnosis. Hydroxyurea was introduced in September 2003 because of WBC count elevation at 36 × 109/l with the presence of a small percentage of promyelocytes, metamyelocytes, and myelocytes, and presence of a splenomegaly. A new chromosomal analysis performed on peripheral blood on November 2003 showed the following karyotype: 46,XX,t(9;22)(q34;q11), t(11;16)(p12;q21)/46,XX. The pronostic Sokal and the Hasford scores at onset of Ph1 chromosome were intermediate at 1.03 and 1087.7, respectively. A treatment with imatinib was started in December 2003 at 400 mg/day allowing stable CML complete hematologic response to date with normalized platelet counts, no splenomegaly, and 5% myelemia. Retrospective interphase Fluorescence in situ hybridisation (FISH) analysis performed with dual color probes for BCR and ABL genes (LSI bcr/abl ES Dual Color Translocation Probe (VYSIS)) on the two bone marrow samples (which had shown a normal karyotype during the first two years of the CMML) did not detect the BCR/ABL fusion gene over 500 interphase nuclei. FISH analysis performed with the same probe at the time of apparition of chromosomal abnormalities showed a typical M-bcr picture (data not shown) in five metaphases and 50 interphase nuclei with 50 normal nuclei.
Reverse transcription (RT) and quantitative real-time polymerase chain reaction (RQ-PCR) were performed from peripheral blood samples according to Standardised EAC (European Against Cancer) protocols previously described9 apart from using the LightCycler technology (Roche Diagnostics, Mannheim, Germany). In this case, no fusion transcript was detected using this approach. In order to detect a rare BCR-ABL junctions, we next used the LightCycler t(9;22) quantification kit (Roche Diagnostics) that contains a mix of two forward primers in exon e1 and b2 from BCR gene and a reverse primer and a probe in exon a4 and a3 from ABL gene, respectively. This multiplex technology allows the detection of all BCR-ABL transcripts and in this way we were able to readily detect the presence of an atypical fragment (Figure 1b), which turned out to correspond to an e6a2 fusion transcript after direct sequencing. The monitoring of the minimal residual disease (MRD) of this case was carried out using real-time PCR with a new e6 forward primer, located close to the e6-bcr in exon 6 (e6: 5′-IndexTermATCCAACGACCAAGAACTCT-3′) from BCR gene and reverse primer (ENR561) and probe (ENP541) from ABL gene.9 Primers (ENF1003, EN R1063) and probe (EN P1043) from ABL control gene were used to normalise results.9 For PCR calibration, a serial 10-fold dilution series for e6a2 cDNA from sample before imatinib mesylate therapy (ranging from 106 to 10 copies) was amplified and the assay was found to be linear over at least five orders of magnitude (slope, −3.5; intercept, 43.2). The final results were calculated as the ratios BCR-ABL/ABL. After 3 months of imatinib mesylate treatment, quantification of the MRD revealed a reduction of the BCR-ABL/ABL ratio (from 0.91 to 0.0089), confirming thus efficacy of this molecule in patients with rare BCR-ABL transcripts. Indeed, a longer follow-up is necessary for proper assessment of residual disease and to see if the clinical outcome of this patient will be also worse as previously suggested.8
A late appearing secondary Philadelphia chromosome along the course of hematopoietic malignancies has been rarely described and is considered as a poor prognostic factor. Furthermore, some authors hypothesize that shorter BCR-ABL transcripts give rise to a more aggressive clinical phenotype and early transformation due to the lack of important regulatory BCR sequences.8 Since very few patients have been reported with this unusual e6a2 BCR-ABL transcript, prediction concerning their clinical outcome can not yet be made. The study of these unusual BCR-ABL junctions and their associated disease seems to be essential to gain more insights into their molecular pathological function and a more comprehensive survey of the different functions of the BCR-ABL chimeric protein.
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This work was supported by the Hospices Civils de Lyon. We thank Annick Bertholin and Christophe Ollagnier for technical assistance.
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Hayette, S., Tigaud, I., Thomas, X. et al. Identification of a rare e6a2 BCR-ABL fusion gene during the disease progression of chronic myelomonocytic leukemia: a case report. Leukemia 18, 1735–1736 (2004). https://doi.org/10.1038/sj.leu.2403476
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