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CML patient with rare b2a3 (e13a3) variant of BCR–ABL transcript: Complete molecular response to imatinib
Leukemia Research 29 (2005) 1365–1366
Letter to the Editor
CML patient with rare b2a3 (e13a3) variant of BCR–ABL transcript: Complete molecular response to imatinib Chronic myeloid leukemia (CML) is characterized by translocation t(9;22)(q34;q11) which results in a BCR–ABL fusion gene, the essential cause of CML pathogenesis. The predominant majority of breakpoints in CML patients occurs in the major breakpoint cluster region of BCR gene (M-BCR) and upstream of a2 exon of ABL gene. Breakpoints outside these two regions occur infrequently. Up to now only about 18 cases with rearrangements eliminating a2 exon of the ABL
gene and forming BCR-a3 fusion transcripts have been described [1–5,8]. Absence of a2 exon partially coding SH3 domain raises the question of whether SH3 plays a special role in CML pathogenesis with impact to the treatment strategy. Up to now contradictory opinions have occurred. Therefore, all these cases should be thoroughly studied. We report another patient with BCR-a3 rearrangement and present molecular monitoring of his response to imatinib by real-time RT-PCR. A 42-year-old man was diagnosed with CML in chronic phase in March 2002. Peripheral blood analysis showed white blood cell (WBC) count 27.8 × l09 /l with 17% mye-
Fig. 1. Molecular, cytogenetic and hematologic responses to the therapy in a patient with b2a3 fusion transcript. Upper panel: WBC, white blood cell count (full line); PLT, platelet count (dotted line). Lower panel: BCR–ABL transcript level (BCR–ABL/B2M × 100) is represented by the line with squares (empty squares, negative results); horizontal lines represent the type of treatment: HU, hydroxyurea; IFN, interferon-alpha; STI, imatinib mesylate, Glivec. The asterisks indicate datapoints of cytogenetic analyses: G, percentage of Ph+ metaphases by conventional Giemsa banding. The numbers of processed metaphases are given in square brackets. F, percentage of BCR–ABL positive interphase nuclei by interphase (I) FISH (LSI BCR–ABL extra signal dual color translocation probe set from ABBOTT-VYSIS; 200 interphase nuclei were screened, the cut-off level of I-FISH analyses was 2.5%). BCR–ABL transcript level was monitored using real-time RT-PCR with non-specific dye SybrGreen I. BCR–ABL was normalized to beta-2-microglobuline (B2M). BCR–ABL primers according to Cross at al. [6], B2M primers according to Pallisgard et al. [7] were used. Close efficiencies were confirmed for BCR–ABL and B2M amplification. Ten BCR–ABL molecules/l could be detected. 0145-2126/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2005.04.001
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Letter to the Editor / Leukemia Research 29 (2005) 1365–1366
locytes, 5% metamyelocytes, 11% bands, 42% segmented neutrophils, 1% basophils, 4% eosinophils, 2% monocytes and 18% lymphocytes; hemoglobin was143 g/l, platelet count 233 × l09 /l. The spleen was not palpable, Socal and Hasford scores were of low risk. Conventional cytogenetic analysis of bone marrow by G-banding demonstrated t(9;22)(q34;q11) in 20/22 metaphases with no additional chromosomal abnormality. Interphase FISH (I-FISH) analysis showed a positive result in 166 of 200 interphases (Fig. 1). Molecular analysis performed using multiplex RT-PCR [6] showed an atypical shorter band, sequencing of which found junction between BCR exon b2 and ABL exon a3, i.e. b2a3 (e13a3) fusion transcript. No minor b2a2 band along with the predominant b2a3 band, as reported by some authors [3,5], was detected even by nested RT-PCR. The patient was rather a poor responder to IFN; no cytogenetic and only partial hematologic response was achieved. However, after the crossover to imatinib, complete hematologic response was attained within 8 weeks and complete cytogenetic remission (CCR) within 7 months. The BCR–ABL monitoring by real-time RT-PCR started only 5 months after the commencement of imatinib therapy but it was performed during a very important period after the achievement of CCR (Fig. 1). In that period, the BCR–ABL showed continual decrease down to real-time PCR negativity and then to two-step RT-PCR negativity at 15 and 18 months on imatinib, respectively. In the interval between 5 and 15 months of imatinib therapy (19–29 months after diagnosis) a 2-log reduction in BCR–ABL transcript level was found. Starting from the time of diagnosis, where the patient’s sample was also available, the total decrease in the BCR–ABL level was of 4 logs. It seems very probable that the total decrease in BCR–ABL level was exclusively caused by imatinib therapy because no cytogenetic response and only partial hematologic response were achieved during IFN treatment. In such cases the decrease in BCR–ABL is not usually detected. Up to now, seven BCR-a3 patients treated with imatinib after IFN pretreatment, including our case, have been reported [1–3,5,8]. Only in our case molecular response was monitored. In most patients cytogenetic examinations were performed, in one patient only hematologic data were given [5] and in one single nested RT-PCR was used [8]. In all cases, where the tests were performed, complete cytogenetic (6/6) or molecular responses (2/2) were detected. It suggests very good responses to imatinib, maybe even better than responses in patients with common BCR–ABL rearrangements. However, more long-term studies, including molecular monitoring preferably with a standardized method, are necessary in order to show the quality and durability of the response. In conclusion, our results together with the published studies suggest that BCR-a3 patients are very good responders to imatinib. As poor responses to IFN are often presented in these patients, imatinib as the first-line therapy seems a very promising treatment strategy.
Acknowledgment The study was supported by grant NC/7550-3 from the Internal Grant Agency of Ministry of Health of the Czech Republic.
References [1] Al-Ali HK, Leiblein S, Kovacs I, Hennig E, Niederwieser D, Deininger MWN. CML with an e1a3 BCR–ABL fusion: rare, benign, and a potential diagnostic pitfall. Blood 2002;100: 1092–3. [2] Liu LG, Tanaka H, Ito K, Kyo T, Ito T, Kimura A. Chronic myelogonous leukemia with e13a3 (b2a3) type of BCR–ABL having a DNA breakpoint between ABL exons a2 and a3. Am J Hematol 2003;74:268–772. [3] Snyder DS, McMahon R, Cohen SR, Slovak ML. Chronic myeloid leukemia with an e13a3 BCR–ABL fusion: benign course responsive to imatinib with an RT-PCR advisory. Am J Hematol 2004;75: 92–5. ˇ [4] Pol´ak J, Zemanov´a Z, Michalov´a K, Klamov´a H, Cerm´ ak J, Haˇskovec C. A new case of chronic myeloid leukemia (CML) in myeloid blast crisis with an atypical (b3/a3) junction of the BCR/ABL gene. Leukemia 1998;12:250. [5] Otaz´u IB, Belen RM, Olicio R, Pinto A, Zalcberg I, Seuanez HN. A rare, in frame BCR–ABL fusion (e13a3) in a patient with an aggressive chronic myeloid leukemia. Acta Haematol 2002;108: 150–3. [6] Cross NCP, Melo JV, Feng L, Goldman JM. An optimized multiplex polymerase chain reaction (PCR) for detection of BCR–ABL fusion mRNAs in hematological disorders. Leukemia 1994;8:186–9. [7] Pallisgaard N, Clausen N, Schroder H, Hokland P. Rapid and sensitive minimal residual disease detection in acute leukemia by quantitative real-time RT-PCR exemplified by t(12;21) TEL-AML1 fusion transcript. Genes Chromosomes Cancer 1999;26:355–65. [8] M¨uller MC, Gattermann N, Lahaye T, Deininger MWN, Berndt A, Fruehauf S, et al. Dynamics of BCR–ABL mRNA expression in firstline therapy of chronic myelogenous leukemia patients with imatinib or interferon ␣/ara-C. Leukemia 2003;17:2392–400.
Jana Moravcov´a a,∗ Jana Rulcov´a a Jaroslav Pol´ak a Hana Klamov´a a Cedrik Haˇskovec a Zuzana Zemanov´a b a Institute of Hematology and Blood Transfusion, Prague, Czech Republic b Center of Oncocytogenetics, Institute of Clinical Biochemistry and Laboratory Diagnosis, General Faculty Hospital and 1st Faculty of Medicine of Charles University, Prague, Czech Republic ∗ Corresponding author. Tel.: +420 221 977 181 fax: +420 221 977 371 E-mail address: [email protected] (J. Moravcov´a) 18 March 2005 Available online 3 May 2005
Letter to the Editor
CML patient with rare b2a3 (e13a3) variant of BCR–ABL transcript: Complete molecular response to imatinib Chronic myeloid leukemia (CML) is characterized by translocation t(9;22)(q34;q11) which results in a BCR–ABL fusion gene, the essential cause of CML pathogenesis. The predominant majority of breakpoints in CML patients occurs in the major breakpoint cluster region of BCR gene (M-BCR) and upstream of a2 exon of ABL gene. Breakpoints outside these two regions occur infrequently. Up to now only about 18 cases with rearrangements eliminating a2 exon of the ABL
gene and forming BCR-a3 fusion transcripts have been described [1–5,8]. Absence of a2 exon partially coding SH3 domain raises the question of whether SH3 plays a special role in CML pathogenesis with impact to the treatment strategy. Up to now contradictory opinions have occurred. Therefore, all these cases should be thoroughly studied. We report another patient with BCR-a3 rearrangement and present molecular monitoring of his response to imatinib by real-time RT-PCR. A 42-year-old man was diagnosed with CML in chronic phase in March 2002. Peripheral blood analysis showed white blood cell (WBC) count 27.8 × l09 /l with 17% mye-
Fig. 1. Molecular, cytogenetic and hematologic responses to the therapy in a patient with b2a3 fusion transcript. Upper panel: WBC, white blood cell count (full line); PLT, platelet count (dotted line). Lower panel: BCR–ABL transcript level (BCR–ABL/B2M × 100) is represented by the line with squares (empty squares, negative results); horizontal lines represent the type of treatment: HU, hydroxyurea; IFN, interferon-alpha; STI, imatinib mesylate, Glivec. The asterisks indicate datapoints of cytogenetic analyses: G, percentage of Ph+ metaphases by conventional Giemsa banding. The numbers of processed metaphases are given in square brackets. F, percentage of BCR–ABL positive interphase nuclei by interphase (I) FISH (LSI BCR–ABL extra signal dual color translocation probe set from ABBOTT-VYSIS; 200 interphase nuclei were screened, the cut-off level of I-FISH analyses was 2.5%). BCR–ABL transcript level was monitored using real-time RT-PCR with non-specific dye SybrGreen I. BCR–ABL was normalized to beta-2-microglobuline (B2M). BCR–ABL primers according to Cross at al. [6], B2M primers according to Pallisgard et al. [7] were used. Close efficiencies were confirmed for BCR–ABL and B2M amplification. Ten BCR–ABL molecules/l could be detected. 0145-2126/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2005.04.001
1366
Letter to the Editor / Leukemia Research 29 (2005) 1365–1366
locytes, 5% metamyelocytes, 11% bands, 42% segmented neutrophils, 1% basophils, 4% eosinophils, 2% monocytes and 18% lymphocytes; hemoglobin was143 g/l, platelet count 233 × l09 /l. The spleen was not palpable, Socal and Hasford scores were of low risk. Conventional cytogenetic analysis of bone marrow by G-banding demonstrated t(9;22)(q34;q11) in 20/22 metaphases with no additional chromosomal abnormality. Interphase FISH (I-FISH) analysis showed a positive result in 166 of 200 interphases (Fig. 1). Molecular analysis performed using multiplex RT-PCR [6] showed an atypical shorter band, sequencing of which found junction between BCR exon b2 and ABL exon a3, i.e. b2a3 (e13a3) fusion transcript. No minor b2a2 band along with the predominant b2a3 band, as reported by some authors [3,5], was detected even by nested RT-PCR. The patient was rather a poor responder to IFN; no cytogenetic and only partial hematologic response was achieved. However, after the crossover to imatinib, complete hematologic response was attained within 8 weeks and complete cytogenetic remission (CCR) within 7 months. The BCR–ABL monitoring by real-time RT-PCR started only 5 months after the commencement of imatinib therapy but it was performed during a very important period after the achievement of CCR (Fig. 1). In that period, the BCR–ABL showed continual decrease down to real-time PCR negativity and then to two-step RT-PCR negativity at 15 and 18 months on imatinib, respectively. In the interval between 5 and 15 months of imatinib therapy (19–29 months after diagnosis) a 2-log reduction in BCR–ABL transcript level was found. Starting from the time of diagnosis, where the patient’s sample was also available, the total decrease in the BCR–ABL level was of 4 logs. It seems very probable that the total decrease in BCR–ABL level was exclusively caused by imatinib therapy because no cytogenetic response and only partial hematologic response were achieved during IFN treatment. In such cases the decrease in BCR–ABL is not usually detected. Up to now, seven BCR-a3 patients treated with imatinib after IFN pretreatment, including our case, have been reported [1–3,5,8]. Only in our case molecular response was monitored. In most patients cytogenetic examinations were performed, in one patient only hematologic data were given [5] and in one single nested RT-PCR was used [8]. In all cases, where the tests were performed, complete cytogenetic (6/6) or molecular responses (2/2) were detected. It suggests very good responses to imatinib, maybe even better than responses in patients with common BCR–ABL rearrangements. However, more long-term studies, including molecular monitoring preferably with a standardized method, are necessary in order to show the quality and durability of the response. In conclusion, our results together with the published studies suggest that BCR-a3 patients are very good responders to imatinib. As poor responses to IFN are often presented in these patients, imatinib as the first-line therapy seems a very promising treatment strategy.
Acknowledgment The study was supported by grant NC/7550-3 from the Internal Grant Agency of Ministry of Health of the Czech Republic.
References [1] Al-Ali HK, Leiblein S, Kovacs I, Hennig E, Niederwieser D, Deininger MWN. CML with an e1a3 BCR–ABL fusion: rare, benign, and a potential diagnostic pitfall. Blood 2002;100: 1092–3. [2] Liu LG, Tanaka H, Ito K, Kyo T, Ito T, Kimura A. Chronic myelogonous leukemia with e13a3 (b2a3) type of BCR–ABL having a DNA breakpoint between ABL exons a2 and a3. Am J Hematol 2003;74:268–772. [3] Snyder DS, McMahon R, Cohen SR, Slovak ML. Chronic myeloid leukemia with an e13a3 BCR–ABL fusion: benign course responsive to imatinib with an RT-PCR advisory. Am J Hematol 2004;75: 92–5. ˇ [4] Pol´ak J, Zemanov´a Z, Michalov´a K, Klamov´a H, Cerm´ ak J, Haˇskovec C. A new case of chronic myeloid leukemia (CML) in myeloid blast crisis with an atypical (b3/a3) junction of the BCR/ABL gene. Leukemia 1998;12:250. [5] Otaz´u IB, Belen RM, Olicio R, Pinto A, Zalcberg I, Seuanez HN. A rare, in frame BCR–ABL fusion (e13a3) in a patient with an aggressive chronic myeloid leukemia. Acta Haematol 2002;108: 150–3. [6] Cross NCP, Melo JV, Feng L, Goldman JM. An optimized multiplex polymerase chain reaction (PCR) for detection of BCR–ABL fusion mRNAs in hematological disorders. Leukemia 1994;8:186–9. [7] Pallisgaard N, Clausen N, Schroder H, Hokland P. Rapid and sensitive minimal residual disease detection in acute leukemia by quantitative real-time RT-PCR exemplified by t(12;21) TEL-AML1 fusion transcript. Genes Chromosomes Cancer 1999;26:355–65. [8] M¨uller MC, Gattermann N, Lahaye T, Deininger MWN, Berndt A, Fruehauf S, et al. Dynamics of BCR–ABL mRNA expression in firstline therapy of chronic myelogenous leukemia patients with imatinib or interferon ␣/ara-C. Leukemia 2003;17:2392–400.
Jana Moravcov´a a,∗ Jana Rulcov´a a Jaroslav Pol´ak a Hana Klamov´a a Cedrik Haˇskovec a Zuzana Zemanov´a b a Institute of Hematology and Blood Transfusion, Prague, Czech Republic b Center of Oncocytogenetics, Institute of Clinical Biochemistry and Laboratory Diagnosis, General Faculty Hospital and 1st Faculty of Medicine of Charles University, Prague, Czech Republic ∗ Corresponding author. Tel.: +420 221 977 181 fax: +420 221 977 371 E-mail address: [email protected] (J. Moravcov´a) 18 March 2005 Available online 3 May 2005