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CORRESPONDENCE The BCR-ABL chimeric gene results from a reciprocal
translocation, t(9;22)(q34;q11). The breakpoint on chromosome 9 is in the majority of cases 5' to ABL exon 2, whereas the
breakpoints on chromosome 22 can occur in various regions within the
BCR gene, giving rise to different BCR-ABL
junction types. Breakpoints within the so-called major BCR
(M-BCR) lead to a BCR-ABL messenger RNA with e13a2 or
e14a2 junction in 99% of patients with chronic myeloid leukemia (CML).
Two other breakpoint types outside the M-BCR have been
described: the m-BCR (e1a2 junction), mainly detected
in Ph+ acute lymphoblastic leukemia, and the µ-BCR
(e19a2 junction) associated with neutrophilic-CML.1-6 We report a novel BCR-ABL fusion transcript with e15a2
junction detected in 2 patients with a presumptive diagnosis of
atypical chronic myeloproliferative disorder.7 Patient 1, a 65-year-old male with an 8-year history of persistent unexplained
neutrophilic leukocytosis (15 × 109/L) was referred to
us in December 1998 with a 5-month history of recurrent fever, weight
loss, fatigue, and anorexia, with no apparent evidence of infectious
diseases. Blood film examination showed 74% neutrophils with no shift
to the left in the granulocytic lineage, 24% lymphocytes, and 2%
monocytes with enlarged platelets. The patient received no treatment
and currently remains asymptomatic with persistent moderate
leukocytosis. Patient 2, a 59-year-old male with a history of diabetes,
hypertension, and alcoholism, was referred to our hospital in September
1998 because of leukocytosis (20 × 109/L) detected in a
routine blood testing. Blood film showed megaloblastic anemia and
neutrophilic leukocytosis with no shift to the left. Serum tests showed
low iron levels and increased ferritin (1000 ng/mL). Bone marrow smear
and biopsy disclosed dysplastic features and maturation defects
of the erythropoietic lineage and a hypogranular granulocytic series.
Neutrophilic leukocytosis persisted with no other relevant associated
symptoms until last control (September 1999) after which the
patient was lost to follow-up. More detailed clinical and laboratory
features of the 2 patients are summarized in Table
1.
Cytogenetic analyses were performed in bone marrow cells and showed in both cases a normal karyotype (46,XY), with no evidence of the Philadelphia chromosome. Molecular analyses were performed on total cellular RNA from peripheral mononuclear blood. RNA isolation, the reverse transcription-polymerase chain reaction (RT-PCR) assay for detection of BCR-ABL transcripts and semiquantitative G3PDH expression (to ensure the efficiency of the reverse-transcription step and to confirm successful amplification of intact complementary DNA) were performed according to previously reported protocols.8-10 In both patients, no PCR products were detected after the first PCR
round. False-negative results were ruled out, due to successful amplification of the control gene.9,10 But an unpredicted product distinct from the already described BCR-ABL fusion
transcripts was detected after nested PCR (with a sensitivity of
10-5 to 10-6 µg of RNA). Direct sequencing of
this fragment revealed a previously unreported BCR-ABL
hybrid originated from an e15a2 junction. In fact, 86 base pairs
of BCR exon 15 were joined in frame with ABL exon
2 at nucleotide 78 (GenBank accession number GI7406986). The
difference between total base pairs gained and lost was 9, or 3 amino
acids (Figure 1). Thus translation of
such an e15a2 transcript would result in a protein that is only 3 amino
acids longer than the classical P210 BCR-ABL, though quite
different in the composition of amino acid residues (Figure
2).
Some rare BCR-ABL fusion types have occasionally been reported in the literature.4 The 2 cases hereby described were identified among 225 patients with myeloproliferative syndromes studied in our laboratory. To the best of our knowledge, these are the first reported cases with a variant BCR-ABL fusion transcript involving BCR exon 15. Interestingly, such molecular aberration was only detected in a minor clone after nested PCR and was associated with mild clinical symptoms (moderate leukocytosis that did not increase with time) and an indolent clinical course. As to the reasons underlying the detection of very low levels of e15a2 transcript in our patients, we believe that the successful amplification of the endogenous G3PDH may rule out the occurrence of technical problems. Two other explanations that may account for this features include (a) the presence of a small subpopulation of BCR-ABL-expressing cells and (b) a low level of BCR-ABL transcript expressed by the entire cell population. In light of the absence of the Philadelphia chromosome after conventional karyotypic analysis, we are more prone to favor the former hypothesis. Recently, the presence of BCR-ABL transcripts only
detectable at the RT-PCR level has been associated with some cases of
Ph In conclusion, the novel BCR-ABL fusion type hereby described and/or its low levels detected might be associated with a phenotype of mild leukocytosis. But more cases showing these molecular and clinical features should be described before such a hypothesis is confirmed. The identification of new transcripts confirms the heterogeneity of breakpoints in BCR-ABL rearrangements. The roles of different BCR-ABL fusion proteins and their relationships to distinct leukemic or indolent phenotypes still deserve further investigation.
María del Pilar Moreno, María Noel Cortinas, Rossana Bonomi, Adriana Cardeza, and María del Rosario Uriarte We thank Drs Francesco Lococo and Gustavo Folle for valuable discussions and critical reading of the manuscript. In addition, we are grateful to Dr Cristina Mogdasy, head of Clinical Laboratory, Asociación Española Primera de Socorros Mutuos, for her support. References 1. Rowley JD. A new consistent chromosomal abnormality in CML identified by quinacrine fluorescence and Giemsa staining. Nature. 1973;243:290-293[CrossRef][Medline] [Order article via Infotrieve].
2.
Hochhaus A, Reiter A, Skladny H, et al.
A novel BCR-ABL fusion gene (e6a2) in a patient with philadelphia chromosome-negative chronic myelogenous leukemia.
Blood.
1996;88:2236-2240
3.
Pane F, Frigeri F, Sindona M, et al.
Neutrophilic-chronic myeloid leukemia: a distinct disease with a specific molecular marker (BCR/ABL with C3/A2 junction).
Blood.
1996;88:2410-2414
4.
Melo JV.
The diversity of BCR-ABL fusion protein and their relationship to leukemia phenotype.
Blood.
1996;88:2375-2384
5.
Mittre H, Leymarie P, Macro M, Leporrier M.
A new case of chronic myeloid leukemia with c3a2 BCR-ABL junction. Is it really a distinct disease?
Blood.
1997;89:4239-4241 6. How GF, Tan LT, Lim LC. Chronic myeloid leukemia with e19a2 (c3a2) BCR/ABL fusion junction-is it truly a benign disease? Leukemia. 1998;12:1166-1167[Medline] [Order article via Infotrieve]. 7. Moreno MP, Cortinas MN, Bonomi R, Uriarte MR. Identification of novel BCR-ABL fusion transcript in two patients with persistent neutrophilia in absence of Philadelphia chromosome. Blood. 1999;94(suppl 1):177b.
8.
Sagayadan GE, Wiernik PH, Sun N, et al.
Effect of retinoic acid and interferon
9.
Da Cheng Z, Hallam SJ, Lee SJ, et al.
Constitutive expression of cellular retinoic acid binding protein II and lack of correlation with sensitivity to all-trans retinoic acid in acute promyelocytic leukemia cells.
Cancer Res.
1998;58:5770-5776 10. Bonomi R, Giordano H, Moreno MP, et al. Simultaneous PML/RAR expression with t(15;17) in onset, and relapse with only t(8;21) in an acute promyelocitic leukemia patient. Cancer Genet Cytogenet. 2000;123:41-43[Medline] [Order article via Infotrieve].
11.
Blickstein D, Aviram A, Luboshitz J, et al.
BCR-ABL transcripts in bone marrow aspirates of Philadelphia negative essential thrombocytopenia patient: clinical presentation.
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Marasca R, Luppi M, Zucchini P, Longo G, Torelli G, Emilia G.
Might essential thrombocythemia carry Ph anomaly?
Blood.
1998;91:3084-3085
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Hackwell S.
Patients with essential thrombocythemia do not express BCR-ABL transcripts.
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1999;93:2420-2421   CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
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| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
essential thrombocytemia,
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