a chronic myeloid leukemia (CML) patient. TO THE EDITOR. The role of the different BCR/ABL fusion proteins in determining the phenotype of the Philadelphia ...
Leukemia (1999) 13, 1463–1472 1999 Stockton Press All rights reserved 0887-6924/99 $15.00 http://www.stockton-press.co.uk/leu
CORRESPONDENCE Concomitant expression of the rare E1/A3 and B2/A3 types of BCR/ABL transcript in a chronic myeloid leukemia (CML) patient TO THE EDITOR The role of the different BCR/ABL fusion proteins in determining the phenotype of the Philadelphia chromosome-positive (Ph-positive) leukemias is still unclear.1 Most chronic myeloid leukemia (CML) patients express BCR/ABL transcripts with a b3/a2 or b2/a2 fusion (see Figure 1) and a translated P210 protein. Approximately two-thirds of the patients with a Ph-positive acute lymphoid leukemia (ALL) express a shortened BCR/ABL protein, called P185, that is translated from a BCR/ABL transcript with a e1a2 type of junction (Figure 1). Only sporadic CML cases expressing exclusively P185 have so far been described, but small amounts of BCR/ABL transcripts with the P185 junction can also be found in CML patients expressing P210 and are believed to arise from a mechanism of alternative splicing of the
longer b2a2 or b3a2 component.2 Finally, a rare breakpoint which produces a BCR/ABL e19a2 junction and predicts a 230 kDa fusion protein, has been identified in patients generally showing a mild form of CML, which has been tentatively denominated Ph-positive CML.3 In all these ‘common’ types of BCR/ABL fusion, the molecular variability is due to a variable breakpoint position within the BCR gene on chromosome 22 and the second exon of the ABL gene (a2) is juxtaposed ‘in frame’ to different portions of the BCR gene. Very rarely, due to a breakpoint position on chromosome 9 within the third intron of the ABL gene, the BCR/ABL fusions show the absence of the ABL exon 2 sequences.4 So far, only three Ph-positive ALL and three CML patients displaying a b2/a3 or b3/a3 junction have been described, whereas expression of the e1/a3 BCR/ABL transcript has been reported only in two pediatric cases of Ph-positive ALL.4 Here we report the
Figure 1 Schematic representation of BCR, ABL, and BCR/ABL mRNA transcripts. The most frequently reported BCR/ABL fusion proteins with their molecular mass (P210, P190 and P200) and their corresponding BCR/ABL RNA type of junctions are indicated on the left side. White boxes, BCR exons not in scale numbered from e1 to e23; grey boxes, ABL exons not in scale numbered from a1 (alternatively spliced exons aIb and aIa, were represented) to a11. Primers AZ, R112, EA12, EA122, and EA500 used for RT-PCR for BCR and BCR/ABL are designated as arrows. The expected amplification product sizes with different combination of primers were designated as a ↔ and the corresponding number of base pairs (bp) is given above the lines.
Correspondence: G Martinelli, Molecular Biology Unit, Institute of Hematology and Medical Oncology ‘Sera`gnoli’, University of Bologna, Via Massarenti, 9, 40138 Bologna, Italy; Fax: 0039 051 398973 Received 3 March 1999; accepted 26 May 1999
finding of a CML patient showing a concomitant expression of both the rare e1/a3 and b2/a3 types of BCR/ABL transcripts, both lacking the ABL exon 2 sequences. A 68-year-old female patient was diagnosed as being in chronicphase CML in June 1997. At that time, her peripheral blood count showed 38.4 × 109/l WBC (88% neutrophils, 1% basophils, 1% pro-
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sequenced and submitted to comparison by FASTA3.6 The two different transcripts, respectively with a b2a3 and e1a3 types of junction, lacked ABL exon 2 sequences. The transcript with the e1a3 junction was probably arising through a mechanism of alternative splicing of the longer form, as a single Ph-chromosome was detectable in all the mitoses obtained from bone marrow cells of the patient. ABL exon 2 encodes for 58 amino acids, the last 17 of which are part of a stretch of 50 amino acids that form the SH3 region of the ABL protein. The SH3 region is thought to negatively regulate the kinase domain (SH1), and its deletion within the BCR/ABL hybrid could be expected, at least in theory, to result in an increased tyrosine-kinase activity and therefore in an increased transforming ability of the corresponding protein. The finding that hybrid BCR/ABL genes lacking the ABL exon 2 sequences were naturally occurring in a small number of patients with Ph-positive leukemias and generally associated with an ALL phenotype or a rapidly progressive CML phenotype could be in favor of this hypothesis. However, here we show that both main BCR/ABL fusion transcripts lacking ABL exon 2 sequences can be simultaneously expressed by the same Ph-positive clone in a patient showing a classical form of CML and this adds further complexity to an enigma that puzzles clinical and molecular hematologists: the relationship existing between the BCR/ABL hybrid gene structure and the leukemia phenotype.
Acknowledgements Figure 2 Agarose gel electrophoresis separation of PCR for BCR/ABL transcripts. Lane 1 (a and b), molecular weight marker VIII from Boehringer Mannheim (Mannheim, Germany) with some sizes of DNA which are indicated by numbers on the left side of the picture. (a) Lanes 2 and 3, RT-PCR with primers EA122-EA500 from total RNA from CML Ph+ patient with b3/a2 and b2/a2 type of transcript, respectively. The expected sizes of PCR products (388 bp and 314 bp, respectively) are given on the left side. Lane 4, RT-PCR from the patient with primers EA122-EA500 showing the presence of an amplified band of 141 bp corresponding to b2/a3 (or e13/a3) type of transcript. Lane 5, negative control. (b) Lane 2, RT-PCR with primers R112-EA500 from total RNA from the patient show the presence of an amplified band of 123 bp corresponding to e1/a3 type of transcript. Lanes 3 and 4, negative controls; lane 5, the T-PCR product from the ALL Ph+ patient amplified with primers R112-EA500 which gave rise to our amplified product of 296 bp (e1/a2 type of transcript); lanes 6 and 7, other RT and PCR negative controls.
myelocytes, 3% lymphocytes, 7% monocytes), 629 × 109/l platelets. The hemoglobin level was 12.9 g/dl, and an alkaline neutrophil phosphatase score of 10 (normal range, 10 to 100). The spleen was not palpable. Bone biopsy showed an increased cellularity of the bone marrow, marked eosinophilia, and normal megakaryocytes. Cytogenetic analysis of 40 bone marrow metaphases by Giemsa banding showed a karyotype with 46XX, t(9;22)(q34;q11). Therapy with ␣interferon (␣-IFN) at 4 MU/m2/day was started: WBC count fluctuated between 2 and 5.5 × 109/l during the following 12 months (hematological remission). Then, due to the progressive loss of hematological remission, ␣-IFN was withdrawn and hydroxyurea was started. At present, she is currently in hematological remission in first chronic phase, after 20 months since diagnosis. The presence of a BCR/ABL transcript was studied at diagnosis by reverse-transcription polymerase chain reaction (RT-PCR).5 Briefly, an amplification product was detected both using primers R112 and AZ (usually positive for e1/a2 transcript in some Ph+ ALL) and using primers EA12 and AZ (usually positive for b2/a2 or b3/a2) transcript): in both reactions the fragments were considerably smaller than expected (approximately 160–170 bp less).5 Specific half-nested RT-PCR with primers EA500 (ABL exon 3), EA122 and with R112 confirmed the amplification of abnormal BCR/ABL transcripts of 141 bp and 123 bp, respectively (Figure 2a and b). Both fragments were directly
This work was supported by Italian Association of Cancer Research (AIRC), by Italian CNR No. 98.00526. CT04 target projects and by ‘30 Ore per la Vita’ AIL grants. The authors are grateful to Mr Robin MT Cooke for editorial assistance and to Chiara Conslovi and Maria Stella Zagarella for technical assistance. G Martinelli1 M Amabile1 C Terragna1 N Testoni1 E Ottaviani1 V Montefusco1 A de Vivo1 M Baccarani2 P Ricci1 G Saglio3 S Tura1
1
Institute of Hematology and Medical Oncology ‘Sera`gnoli’, University of Bologna; 2 Cattedra di Ematologia, University of Udine; and 3 Department of Biomedical Sciences and Human Oncology, University of Turin, Italy
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