Chronic Myelogenous Leukemia with e13a3 (b2a3) Type of BCR-ABL Transcript Having a DNA Breakpoint between ABL exons a2 and a3

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1 American Journal of Hematology 74: (2003) Chronic Myelogenous Leukemia with e13a3 (b2a3) Type of BCR-ABL Transcript Having a DNA Breakpoint between ABL exons a2 and a3 Li-Gen Liu, 1,2 Hideo Tanaka, 1 * Kinro Ito, 1 Taiichi Kyo, 3 Takuo Ito, 1 and Akiro Kimura 1 1 Department of Hematology & Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan 2 Hematological Department, Shanghai Fifth People s Hospital affiliated with Fudan University, Shanghai, China 3 Fourth Department of Internal Medicine, Hiroshima Red Cross & Atomic Bomb Survivors Hospital, Hiroshima, Japan We describe a patient with chronic myelogenous leukemia (CML), in whom the DNA breakpoint in the BCR-ABL fusion gene was determined to result in a rare e13a3 (b2a3) transcript. The breakpoint in BCR was intron 13, which was 30 bp downstream from exon 13, and the breakpoint in ABL was intron 2, and was 46 bp downstream from exon a2. This case conforms to the mechanism of DNA breakage occurring within ABL intron 2, but not at 5 0 to ABL exon a2. With our review of this case and the literature, it seems that CML with the BCR-a3 fusion product is associated with a low proportion of circulating immature cells, mild or lack of splenomegaly, slow progressiveness, rather resistance to IFN-a, and good response to imatinib mesylate. This is the first report of BCR-a3-type CML in which the exact DNA breakpoint was identified and located between exons a2 and a3 of the ABL gene. Am. J. Hematol. 74: , ª 2003 Wiley-Liss, Inc. Key words: chronic myelogenous leukemia; BCR; ABL; DNA breakpoint INTRODUCTION ª 2003 Wiley-Liss, Inc. The hallmark of chronic myelogenous leukemia (CML) is the chimeric BCR-ABL fusion gene, which is usually formed as a result of the t(9;22) translocation (Philadelphia chromosome, Ph). Most BCR-ABL fusion transcripts are e13a2 (b2a2), e14a2 (b3a2), and less commonly, e1a2, e19a2 [1]. CML with an atypical hybrid transcript, in which BCR sequences are fused to ABL exon a3 rather than exon a2, is very rare, and its characteristics are poorly understood [2 11]. Until now, the position of the DNA breakpoint in the ABL gene with BCR-a3-type CML has been reported in only three cases. ABL exon a2 encodes 58 amino acids, the last 17 of which form part of a stretch of 50 amino acids of Src homology 3 (SH3) domain of the ABL protein. The SH3 is believed to negatively regulate the kinase domain (SH1). Thus, in theory, deletion of ABL exon a2 should result in increased tyrosine kinase activity and, therefore, increased transforming activity. However, recent data suggest that SH3 does not necessarily contribute to aggressive phenotype. Details of the clinical phenotype of CML with this type of transcript are unclear because of its very low incidence. Here, we report a case of CML with the e13a3 transcript and the DNA breakpoints in the BCR and ABL genes. This case was rather resistant to interferon a (IFN-a) and showed rapid response to imatinib mesylate (imatinib). This is the first report of the exact DNA breakpoint in the ABL gene determined by nucleotide sequencing in BCR-a3 type CML. Contract grant sponsor: Tsuchiya Foundation *Correspondence to: Hideo Tanaka, M.D., Ph.D., Department of Hematology & Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima , Japan. dtanaka@hiroshima-u.ac.jp Received for publication 21 October 2002; Accepted 15 July 2003 Published online in Wiley InterScience ( DOI: /ajh.10429

2 Case Report: CML Lacking ABL exon a2 269 MATERIALS AND METHODS RNA and DNA were extracted from the cells of a bone marrow aspirate from a patient with CML before treatment. Informed and written consent was obtained before bone marrow aspiration. Reverse transcription polymerase chain reaction (RT-PCR) for specific fusion transcript of major BCR-ABL was performed using sets of primers for sequences within BCR exon 13 and ABL exon a3. The primer sequences were as follows (The primer sequences were derived from GenBank accession numbers U07000 and U07563): BCR1: 5 0 -GCTTCTCCCTGACATCCGTG-3 0 ABL1: 5 0 -GGCCCATGGTACCAGGAGTG-3 0 BCR2: 5 0 -GGAGCTGCAGATGCTGACCAAC-3 0 ABL2: 5 0 -GTTTCTCCAGACTGTTGACTG-3 0 Nested PCR was performed with the set of primers of first, BCR1 and ABL1, and second, BCR2 and ABL2. Genomic DNA PCR was also performed with the same sets of primers. The PCR product was cloned into a vector using TA cloning kit (Invitrogen Corp, Carlsbad, CA) and followed by sequencing in ABI PRISM 310 genetic analyzer (Perkin Elmer, Foster City, CA). normalized (46, XY [20/20]), and FISH for BCR-ABL dropped to 0%, indicating complete cytogenetic response (CCR). RT-PCR was performed to detect BCR-ABL transcript. The product (Fig. 1A, lane 2) showed one band that was smaller than the common e13a2 (lanes 3) or e14a2 (lane 4) transcripts. Sequencing of the PCR product revealed the e13a3-type BCR-ABL transcript, which was 174 bp shorter than that of the usual e13a2 type, meaning that the ABL exon a2 transcript was completely gone. To determine the DNA breakpoints of the BCR- ABL fusion gene, genomic DNA was amplified with the preceding sets of primers. The PCR product showed one band (lane 5) at the position of approximately 800 bp. Sequencing of the product revealed that the DNA breakpoint of BCR was in intron 13, 30 bp downstream from exon 13, and that the breakpoint of ABL was in intron 2 and was 46 bp downstream from exon a2 (Fig. 1B). CASE A 49-year-old man was diagnosed with chronicphase (CP) CML. He had mild splenomegaly at diagnosis in August of. Peripheral blood analysis results were as follows: white blood cell (WBC) count, /L; hemoglobin, 11.5 g/dl; platelet count, /L. The WBC differential count was as follows: 0.5% blasts; 3.5% promyelocytes; 20.5% myelocytes; 11% metamyelocytes; 16.5% band neutrophils; 29.5% segmented neutrophils; 6.0% basophils; 4.5% eosinophils; 1.5% monocytes; 7.0% lymphocytes. The bone marrow aspirate showed hypercellularity, and the karyotype was 46, XY, t(9;22)(q34;q11) [20/20]. The positive rate of BCR-ABL fusion in peripheral blood was 97.2% as determined by fluorescence in situ hybridization (FISH). Starting in September, the patient was treated with IFN-a and hydroxyurea. The peripheral WBC count was /L in March 2001, but the abnormal white blood cell differential (1% promyelocyte, 17% myelocyte, and 10% metamyelocyte) indicated only a partial hematologic response (PHR). Chromosome analysis in August 2001 showed that Ph-positive clone remained at 95%, indicating poor response. FISH analysis at November showed that the positive rate of the BCR-ABL fusion gene was 75.8%. Thus, IFN-a with hydroxyurea did not induce good response. Then, from December 2001, imatinib was administered. In April 2002, the karyotype was Fig. 1. (A) Agarose gel electrophoresis of RT-PCR product of BCR-ABL mrna transcripts and DNA PCR product of BCR- ABL fusion gene from genomic DNA. Lane 1, 1-kb DNA ladder marker.lane2,rt-pcrproductofourpatient sbcr-abl transcript. Lane 3, RT-PCR product of e13a2 (b2a2) as a positive control. Lane 4, RT-PCR product of e14a2 (b3a2) as a positive control. Lane 5, DNA PCR product of BCR-ABL fusion gene from our patient s genomic DNA. (B) Sequencing result of the PCR product from genomic DNA of our patient. The vertical arrow indicates the DNA breakpoint and BCR-ABL junction of the fusion gene. [Color figure can be viewed in the online issue, which is available at

3 270 Case Report: Liu et al. DISCUSSION To our knowledge, this is the first report of CML with the BCR-a3 type, in which the exact DNA breakpoint of the ABL gene was identified. We found that the breakpoint of the ABL gene was between exons a2 and a3. This is consistent with earlier reports of two cases of Ph-positive acute lymphocytic leukemia (ALL) [12] and 1 case of CML with the e13a3 transcript [2]. However, in these cases, the breakpoints were determined by Southern blotting. DNA breakpoint determined by sequencing was reported only in one of those two patients with ALL [12], in which the breakpoint was in ABL intron 2 just like our case. Their breakpoint was 22 bp downstream from exon a2, at a position 24 bp upstream from the breakpoint that we identified in our case. In contrast, there has been one detailed report and one short report of CML patients with the BCRa3-type, in which no DNA rearrangement was detected in ABL intron 2 by Southern blotting, suggesting that deletion of exon a2 from the transcript was due to the splicing mechanism in these cases [3,4]. Thus, at least two different mechanisms seem to be involved in the formation of the BCR-a3 fusion transcript, one is DNA breakage within ABL intron 2, and the other is DNA breakage at 5 0 to ABL exon a2 and thereafter transcript for exon a2 is spliced out. The primary factors that determine preferential breakage sites in the BCR and ABL genes are presently unknown. The low incidence of BCR-a3 fusion product may be due to the relatively short length of ABL intron 2 (0.6 kb) compared with the introns at the beginning of the ABL gene (>200 kb). The theoretically predicted frequency of occurrence of a DNA breakpoint between ABL exons a2 and a3 is 0.3% of BCR-ABL rearranged patients, assuming that breakpoints in ABL are randomly distributed. This theoretical frequency may hold true, because we found this case during the RT-PCR study of 110 CML cases. Alu element is thought to be a strong candidate involved in DNA breakage and chimeric gene rearrangement, because it is the most abundant repeat sequence in both of these genes. However, in our case, we could not find Alu element at the breakpoints of both BCR and ABL genes. A lack of Alu elements was observed across the major and minor breakpoint cluster regions of BCR and across a 25-kb region with a high frequency of breakage in ABL1 [13]. Thus, the incidence and DNA breakage of BCR-a3 type may not be determined only by the size of introns or by Alu element. Thus, mechanisms of DNA breakage in BCR-ABL gene seem more complicated. A review of the literature revealed 13 cases of BCRa3 type CML including our case and 6 cases of BCRa3 type ALL. The clinical data of BCR-a3 type CML are summarized in Table I. There does not seem to be any particular feature with regard to age or gender distribution, WBC count, or platelet count. However, there seemed to be an association with low proportion of circulating immature cells and mild or no splenomegaly. All but 3 of the 13 reported cases had a benign prognosis. The longest lived patient has survived for at least 126 months and was in CP at the last follow-up. Two patients did not require any treatment and remained in the CP for at least 60 and 66 months. This supports the notion that BCR-a3 type CML patients may have a benign prognosis. In eight cases, IFN-a has been administered. Among the five cases in which hematologic responses were assessed, our case achieved PHR, two cases achieved complete hematologic response (CHR), and two cases achieved remission without precise statements from points of response criteria. However, three of these latter four cases lost responsiveness to IFN-a within 2 years. Among the four cases in which cytogenetic responses were assessed, two showed partial cytogenetic response (PCR), one showed no cytogenetic response (NCR), and our case showed MCR, but the Ph chromosome was 95% positive, meaning close to NCR. Imatinib was used only in three cases including our case. All of them achieved CCR. Thus, imatinib treatment seems very promising even for BCR-a3 type CML, in addition to common BCR-a2 type CML. A CML patient with BCR-a3 transcript is a nice model in which to investigate the roles of SH3 domain in BCR-ABL protein in vivo. Bcr-Abl mutants lacking SH3 resulted in reduced tissue invasiveness, and proliferation of leukemic cells slowed down in vivo [14]. Another study demonstrated that both SH3 and SH2 are required for STAT5 activation by BCR-ABL protein [15]. In murine models of CML, e14a3-type of Bcr- Abl/p210 induced CML-like myeloproliferative diseases but showed small delay of the increase in peripheral blood WBC counts and showed small, but consistent, increase in survival compared with the common e14a2- type [16]. These findings seem to agree with the generally less progressive clinical histories of BCR-a3 type CML patients. In conclusion, CML with the BCR-a3 transcript seems less progressive but rather resistant to IFN-a and sensitive to imatinib as is seen in the common BCR-a2 type. Therefore, diagnosis of this type of CML is important, because it may have a different clinical expression and may affect treatment strategies. ACKNOWLEDGMENTS We thank Ryoko Yamaguchi, Sachiko Hidani, and Nanae Nakaju for their excellent technical support.

4 Case Report: CML Lacking ABL exon a2 271 TABLE I. Clinical and Laboratory Characteristics of CML Patients with BCR-ABL Transcripts Lacking ABL Exon a2 Case No Gender Male Male Female Female Male Male NA Male Female Male Female Male Male Age (y) NA Diagnostic phase CP CP CP CP CP CP CP CP CP CP CP CP CP Splenomegaly Mild Yes No No No No NA No No NA No NA Yes Karyotype Ph Ph Ph Ph t(4:9:22) Ph Ph Ph Ph Ph Ph/13q Ph Ph BCR-ABL transcript e13a3 e1a3/e1a2 e1a3 e1a3 e14a3 e14a3 e13a3 e14a3 e1a3/e13a3 e14a3 e14a3 NA e13a3 Hemoglobin level (g/dl) 11.5 NA NA NA NA NA NA (6.0 mmol/l) WBC count (10 9 /L) NA NA 254 WBC differentiation NA NA NA Blast (%) Promyelocyte (%) Myelocyte (%) Metamyelocyte (%) a 22 a Band + segmented (%) Basophils (%) Eosinophils (%) Monocytes (%) Lymphocytes (%) Normoblasts (%) 3 Platelet count (10 9 /L) NA NA NA 180 Initial treatment IFN/Hy IFN/Hy IFN/Hy No No!Hy IFN NA IFN/AraC IFN IFN/Hy No Bu/IFN Hy IFN response (hematologic) IFN response (cytogenetic) PR CR NA NA Remission! MR NR PR PR Remission! CR! Second-line treatment Imatinib Imatinib Imatinib Bu/6MP AutoBMT Hy BMT Imatinib response CR CR CR (cytogenetic) Duration of CP before imatinib (mos) Duration of follow-up* (mos) Clinical outcome CP CP CP CP CP CP CP CP BC CP BC AP Intron 2 DNA breakpoint of ABL gene Authors, published year Intron 2 Upstream of a2 Present case Al-Ali, 2002 Al-Ali, 2002 Roman, 2001 Tribelli, Tribelli, Wilson, Amabile, 1999 Martinelli, 1999 Polák, 1998 Iwata, 1994 Upstream of a2 Páldi- Haris, 1994 van der Plas, 1991 *Between the diagnosis and the last follow-up. a All immature neutrophils included. NA, not applicable; CP, chronic phase; AP, accelerated phase; BC, blastic crisis; Ph, t(9:22)(q34:q11); IFN, interferon; Hy, hydroxyurea; Bu, busulfan; 6-MP, 6-mercaptopurine: auto, autologous; BMT, bone marrow transplantation; AraC, cytarabine; CR, complete response; PR, partial response; MR, minor response; NR, no response (by criteria of Talpaz).

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