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ABL localization on the 9q34 band and duplication of the der(9) in a case of chronic myelogenous leukemia
Cancer Genetics and Cytogenetics 163 (2005) 173–175
Short communication
Masked Philadelphia chromosome due to atypical BCR/ABL localization on the 9q34 band and duplication of the der(9) in a case of chronic myelogenous leukemia Giuseppina Fugazza, Anna Garuti, Stefania Marchelli, Maurizio Miglino, Roberto Bruzzone, Anna Maria Gatti, Sandra Castello, Mario Sessarego* Dipartimento di Medicina Interna e Dipartimento di Ematologia e Oncologia, Universita` degli Studi di Genova, Viale Benedetto XV/6, 16132 Genova, Italy Received 3 March 2005; received in revised form 10 May 2005; accepted 17 May 2005
Abstract
The cytogenetic studies and molecular evaluation of a Philadelphia chromosome negative chronic myelogenous leukemia patient with trisomy 21 (100% metaphases) and trisomy 9 (50% metaphases) at diagnosis are described. Fluorescence in situ hybridization revealed an atypical location of the BCR/ABL fusion signal on 9q, which was duplicated in cells with trisomy 9 simulating a double Ph. The patient was successfully treated with Glivec (also known as Gleevec; Novartis, Basel, Switzerland) and achieved complete hematological and cytogenetic response as well as a reduction of BCR/ABL transcripts detected by real-time quantitative PCR. Ó 2005 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
Chronic myelogenous leukemia (CML) is characterized by the formation of the BCR/ABL chimeric gene, which encodes for atypical tyrosine kinase proteins responsible for transformation to leukemia. The BCR/ABL hybrid gene usually maps to chromosome 22, the Philadelphia chromosome (Ph), which is recognizable because of its small size. In some patients with apparently normal karyotypes, the ABL gene, which usually maps to 9q34, is inserted into the 22q11 region where the BCR gene is located. This gives rise to the BCR/ABL hybrid gene, as it occurs in cases with the standard t(9;22), but without modifying the chromosome morphology [1]. These rare cases can be identified by fluorescence in situ hybridization (FISH), which shows the juxtaposition of ABL to BCR sequences on a cytogenetically ‘‘normal’’ chromosome 22 [2]. The diagnosis of CML can be confirmed using qualitative and/or quantitative polymerase chain reaction (PCR) which reveals the presence of the BCR/ABL transcript [3,4].
We report a 62-year-old male with CML in chronic phase with a hyperdiploid Ph-negative karyotype showing trisomy 21 (100% of metaphases) and trisomy 9 (50% of metaphases) (Fig. 1). The LSI BCR/ABL dual-color, single-fusion translocation probe (S-FISH) and the LSI BCR/ABL dual-color, dual-fusion translocation probe (D-FISH) (Vysis Inc., Downers Grove, IL) demonstrated the presence of the BCR/ABL fusion signal on the distal long arm of a chromosome 9 in all metaphases examined. D-FISH revealed a residual BCR signal on the der(22) (Fig. 2), which is not visible with the S-FISH. Furthermore, the chromosome carrying the BCR/ABL gene was duplicated in the cells containing the trisomy 9. The diagnosis of CML was confirmed by qualitative polymerase chain reaction (PCR), which demonstrated the presence of a typical b3a2 configuration, and by real-time quantitative PCR (RT-Q-PCR) which revealed the presence of a high BCR/ABL transcript value. The patient was treated with Glivec 400 mg/die and achieved a rapid hematologic response. The RT-Q-PCR was performed on peripheral blood samples at the same time as cytogenetic studies using a TAQ-Man system (ABI Prism 7700; Perkin Elmer, Monza, Italy) as previously described by Gabert et al. [5]. The transcript copy numbers were normalized to the
* Corresponding author. Tel.: 0039-010353-7962; fax: 0039-0103538638. E-mail address: [email protected] (M. Sessarego). 0165-4608/05/$ – see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2005.05.010
174
G. Fugazza et al. / Cancer Genetics and Cytogenetics 163 (2005) 173–175
Fig. 1. Q-banded metaphase of a bone marrow metaphase cell showing trisomy 9 (arrows) and trisomy 21 (arrowheads).
number of endogenous ABL transcripts using the following formula: (BCR-ABL/ABL) 10,000 . In agreement with Kaeda et al. [6], we found that a (BCR-ABL/ABL) 10,000 ratio of 2 does not satisfy the criteria for molecular relapse. Conversely, a (BCR-ABL/ABL) 10,000 ratio $5 was sufficient to satisfy the criteria for molecular relapse. Ratio values between 2 and 5 were considered borderline. In order to evaluate the response to Glivec therapy (Novartis, Basel, Switzerland) we used S-FISH and considered a score with more than 5% of cells with fusion signal above background limits indicative for the presence of the Ph. This value was obtained by testing the probe on bone marrow (BM) cells obtained from patients with Ph-negative disorders: the fusion signal positive median value plus 3 times the standard deviation proved to be 4.84% [7]. BM examination, cytogenetic and FISH analysis, and RT-QPCR were performed at 3, 6, and 12 months after starting Glivec treatment.
3. Results and discussion Table 1 summarizes the cytogenetic, FISH, and molecular results obtained after 1 year of follow-up. In our patient, the banding technique did not reveal the standard Ph, but
Fig. 2. FISH on a bone marrow metaphase cell using BCR-ABL dualcolor, dual-fusion probe. Arrow indicates the residual BCR probe signal on the derivative 22.
rather 2 apparently not specific numerical abnormalities, whereas D-FISH showed 1 or 2 BCR/ABL fusion signals, located on 9q34 instead of on 22q11, as usually occurs in standard Ph1 patients and in the rare Ph-negative, BCR/ ABL positive patients [8]. We can hypothesize that such a finding may be due to a second translocation involving the same chromosomes 9 and 22 that originally gave rise to the standard t(9;22), reconstituting the 2 chromosomes, which then appear cytogenetically normal [9]. Another explanation could be an insertion of BCR sequences (5’compared to the major breakpoint cluster region) into the ABL region (5’compared to exon 2). Such insertion could lead to the formation of the classical BCR/ ABL fusion gene that is responsible for the production of the p210 fusion protein. Moreover, FISH revealed that the cells with trisomy 9 showed a duplication of the chromosome 9 that carries the BCR/ABL fusion gene: this must be considered a Ph duplication. Similar observations were reported by Takahashi et al. [10]. It is usually assumed that the duplication of the Ph, which is frequently observed during disease progression, is due to non-disjunction, an event
Table 1 Cytogenetic, FISH, and molecular results at diagnosis and after 3, 6, and 12 months of GlivecÒ therapy Technique
Diagnosis
3 months
6 months
12 months
Banding
47,XY,121[15]/ 48,XY,19,121[16] 95%
46,XY [20]
46,XY [20]
46,XY [20]
8%
4%
4%
7,584
12.58
8.55
3.5
FISH [BCR/ABL fusion signal positive cells]a RT-Q-PCRb
The numbers in brackets refer to the number of observed metaphases. It is worth noting the differences in sensitivity in revealing the progressive reduction of the leukemic clone by banding, FISH, and RT-PCR. a Percentage of positive cells ! 5 is indicative of the absence of Ph. b Ratio ! 5 is considered borderline.
G. Fugazza et al. / Cancer Genetics and Cytogenetics 163 (2005) 173–175
that is still unclear [11]. Keeping in mind that trisomy 9 is a rare event in CML and in the advanced phases of disease as well, we can hypothesize that the duplication of this chromosome is favored by the presence of the BCR/ABL sequence on it. On the other hand, duplication of the derivative chromosome carrying the new gene that is responsible for the disease is not a rare event in leukemia [12–14]. D-FISH is usually performed to demonstrate the presence of a second fusion signal caused by the chimeric ABL/BCR gene on 9q34 [15], or if this signal is absent, to assess deletions of ABL sequences flanking the breakpoint region. This observation has acquired important prognostic significance [16]. In our patient with masked Ph, this prognostic value cannot be documented because the BCR/ABL signal is mapped exactly on 9q34. Lastly, we can state that therapy with Glivec proved to be efficient from a clinical, cytogenetic, and molecular point of view. In fact, RT-Q-PCR demonstrated that the quantitative measurement of the p210 fusion protein decreased from 7,584 before therapy to 12.58 after 3 months, to 8.54 after 6 months, and to 3.5 after 12 months of therapy with Glivec. In other words, despite the unusual cytogenetic features, therapy with Glivec (Novartis, Basel, Switzerland) led to complete and rapid hematological response and to complete cytogenetic response after 3 months with persisting BCR/ ABL positive cells, as revealed by the FISH and RT-QPCR techniques. After 6 months, FISH was negative, and the RT-Q-PCR became weakly positive. Lastly, after 1 year, the RT-Q-PCR value was !5 (borderline value). The unusual location of BCR/ABL sequences on a chromosome 9 and the duplication of the this chromosome apparently did not confer drug resistance.
[5]
[6]
[7]
[8]
[9]
[10]
[11] [12]
[13]
[14]
References [15] [1] Barnes DJ, Melo JV. Cytogenetic and molecular genetic aspects of chronic myeloid leukaemia. Acta Haematol 2002;108:180–202. [2] Bentz M, Cabot G, Moos M, Speicher MR, Ganser A, Lichter P, Dohner H. Detection of chimeric BCR-ABL genes on bone marrow samples and blood smears in chronic myeloid and acute lymphoblastic leukemia by in situ hybridization. Blood 1994;83:1922–8. [3] Wells SJ, Phillips CN, Winton EF, Farhi DC. Reverse transcriptasepolymerase chain reaction for bcr/abl fusion in chronic myelogenous leukemia. Am J Clin Pathol 1996;105:756–60. [4] Amabile M, Giannini B, Testoni N, Montefusco V, Rosti G, Zardini C, Terragna C, Buonamici S, Ottaviani E, Soverini S, Fiacchini M, Bassi S, de Vivo A, Trabacchi E, Saglio G, Pane F, Baccarani M, Tura S, Martinelli G. Real-time quantification of
[16]
175
different types of bcr-abl transcript in chronic myeloid leukemia. Haematologica 2001;86:252–9. Gabert J, Beillard E, van der Velden VH, Bi W, Grimwade D, Pallisgaard N, Barbany G, Cazzaniga G, Cayuela JM, Cave H, Pane F, Aerts JL, De Micheli D, Thirion X, Pradel V, Gonzalez M, Viehmann S, Malec M, Saglio G, van Dongen JJ. Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukaemia. A Europe Against Cancer Program. Leukemia 2003;17:2318–57. Kaeda J, Chase A, Goldman JM. Cytogenetic and molecular monitoring of residual disease in chronic myeloid leukaemia. Acta Haematol 2002;107:64–75. Fugazza G, Miglino M, Bruzzone R, Quintino S, Gatti AM, Grasso R, Gobbi M, Frassoni F, Sessarego M. Cytogenetic and fluorescence in situ hybridization monitoring in Ph1 chronic myeloid leukemia patients treated with imatinib mesylate. J Exp Clin Cancer Res 2004;23:295–9. Lazaridou A, Chase A, Melo J, Garicochea B, Diamond J, Goldman J. Lack of reciprocal translocation in BCR-ABL positive Ph-negative chronic myeloid leukemia. Leukemia 1994;8:454–7. Hagemeijer A, Buijs A, Smit E, Janssen B, Creemers GJ, Van der Plas D, Grosveld G. Translocation of BCR to chromosome 9: a new cytogenetic variant detected by FISH in two Ph-negative, BCR-positive patients with chronic myeloid leukemia. Genes Chromosomes Cancer 1993;8:237–45. Takahashi N, Miura I, Ohshima A, Utsumi S, Nimura T, Hashimoto K, Saito M, Miura AB. Duplication of chromosome 9 carrying a BCR/ABL chimeric gene in Philadelphia chromosome negative chronic myeloid leukemia. Cancer Genet Cytogenet 1996;89: 166–9. Lamb NE, Hassold TJ. Nondisjunction–a view from ringside. N Engl J Med 2004;351:1931–4. Auger MJ, Ross FM, Mackie MJ. 8;21 translocation with duplication of the der(21) in a patient with myelomonocytic leukemia. Cancer Genet Cytogenet 1991;51:139–41. Fugazza G, Cerri R, Bruzzone R, Patrone F, Sessarego M. Duplication of the der(13)t(12;13)(p13;q14) in chronic myelomonocytic leukemia. Haematologica 1997;82:336–7. Maserati E, Casali M, Invernizzi R, Brugnatelli S. Duplication of the der(14) chromosome of a translocation (8;14) in a case of Burkitt’s type L3-ALL. Cancer Genet Cytogenet 1992;61:218–9. Dewald G, Stallard R, Alsaadi A, Arnold S, Blough R, Ceperich TM, Rafael Elejalde B, Fink J, Higgins JV, Higgins RR, Hoeltge GA, Hsu WT, Johnson EB, Kronberger D, McCorquodale DJ, Meisner LF, Micale MA, Oseth L, Payne JS, Schwartz S, Sheldon S, Sophian A, Storto P, Van Tuinen P, Wenger GD, Wiktor A, Willis LA, Yung JF, Zenger-Hain J. A multicenter investigation with D-FISH BCR/ABL1 probes. Cancer Genet Cytogenet 2000;116:97–104. Huntly BJ, Reid AG, Bench AJ, Campbell LJ, Telford N, Shepherd P, Szer J, Prince HM, Turner P, Grace C, Nacheva EP, Green AR. Deletions of the derivative chromosome 9 occur at the time of the Philadelphia translocation and provide a powerful and independent prognostic indicator in chronic myeloid leukemia. Blood 2001;98: 1732–8.
Short communication
Masked Philadelphia chromosome due to atypical BCR/ABL localization on the 9q34 band and duplication of the der(9) in a case of chronic myelogenous leukemia Giuseppina Fugazza, Anna Garuti, Stefania Marchelli, Maurizio Miglino, Roberto Bruzzone, Anna Maria Gatti, Sandra Castello, Mario Sessarego* Dipartimento di Medicina Interna e Dipartimento di Ematologia e Oncologia, Universita` degli Studi di Genova, Viale Benedetto XV/6, 16132 Genova, Italy Received 3 March 2005; received in revised form 10 May 2005; accepted 17 May 2005
Abstract
The cytogenetic studies and molecular evaluation of a Philadelphia chromosome negative chronic myelogenous leukemia patient with trisomy 21 (100% metaphases) and trisomy 9 (50% metaphases) at diagnosis are described. Fluorescence in situ hybridization revealed an atypical location of the BCR/ABL fusion signal on 9q, which was duplicated in cells with trisomy 9 simulating a double Ph. The patient was successfully treated with Glivec (also known as Gleevec; Novartis, Basel, Switzerland) and achieved complete hematological and cytogenetic response as well as a reduction of BCR/ABL transcripts detected by real-time quantitative PCR. Ó 2005 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
Chronic myelogenous leukemia (CML) is characterized by the formation of the BCR/ABL chimeric gene, which encodes for atypical tyrosine kinase proteins responsible for transformation to leukemia. The BCR/ABL hybrid gene usually maps to chromosome 22, the Philadelphia chromosome (Ph), which is recognizable because of its small size. In some patients with apparently normal karyotypes, the ABL gene, which usually maps to 9q34, is inserted into the 22q11 region where the BCR gene is located. This gives rise to the BCR/ABL hybrid gene, as it occurs in cases with the standard t(9;22), but without modifying the chromosome morphology [1]. These rare cases can be identified by fluorescence in situ hybridization (FISH), which shows the juxtaposition of ABL to BCR sequences on a cytogenetically ‘‘normal’’ chromosome 22 [2]. The diagnosis of CML can be confirmed using qualitative and/or quantitative polymerase chain reaction (PCR) which reveals the presence of the BCR/ABL transcript [3,4].
We report a 62-year-old male with CML in chronic phase with a hyperdiploid Ph-negative karyotype showing trisomy 21 (100% of metaphases) and trisomy 9 (50% of metaphases) (Fig. 1). The LSI BCR/ABL dual-color, single-fusion translocation probe (S-FISH) and the LSI BCR/ABL dual-color, dual-fusion translocation probe (D-FISH) (Vysis Inc., Downers Grove, IL) demonstrated the presence of the BCR/ABL fusion signal on the distal long arm of a chromosome 9 in all metaphases examined. D-FISH revealed a residual BCR signal on the der(22) (Fig. 2), which is not visible with the S-FISH. Furthermore, the chromosome carrying the BCR/ABL gene was duplicated in the cells containing the trisomy 9. The diagnosis of CML was confirmed by qualitative polymerase chain reaction (PCR), which demonstrated the presence of a typical b3a2 configuration, and by real-time quantitative PCR (RT-Q-PCR) which revealed the presence of a high BCR/ABL transcript value. The patient was treated with Glivec 400 mg/die and achieved a rapid hematologic response. The RT-Q-PCR was performed on peripheral blood samples at the same time as cytogenetic studies using a TAQ-Man system (ABI Prism 7700; Perkin Elmer, Monza, Italy) as previously described by Gabert et al. [5]. The transcript copy numbers were normalized to the
* Corresponding author. Tel.: 0039-010353-7962; fax: 0039-0103538638. E-mail address: [email protected] (M. Sessarego). 0165-4608/05/$ – see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2005.05.010
174
G. Fugazza et al. / Cancer Genetics and Cytogenetics 163 (2005) 173–175
Fig. 1. Q-banded metaphase of a bone marrow metaphase cell showing trisomy 9 (arrows) and trisomy 21 (arrowheads).
number of endogenous ABL transcripts using the following formula: (BCR-ABL/ABL) 10,000 . In agreement with Kaeda et al. [6], we found that a (BCR-ABL/ABL) 10,000 ratio of 2 does not satisfy the criteria for molecular relapse. Conversely, a (BCR-ABL/ABL) 10,000 ratio $5 was sufficient to satisfy the criteria for molecular relapse. Ratio values between 2 and 5 were considered borderline. In order to evaluate the response to Glivec therapy (Novartis, Basel, Switzerland) we used S-FISH and considered a score with more than 5% of cells with fusion signal above background limits indicative for the presence of the Ph. This value was obtained by testing the probe on bone marrow (BM) cells obtained from patients with Ph-negative disorders: the fusion signal positive median value plus 3 times the standard deviation proved to be 4.84% [7]. BM examination, cytogenetic and FISH analysis, and RT-QPCR were performed at 3, 6, and 12 months after starting Glivec treatment.
3. Results and discussion Table 1 summarizes the cytogenetic, FISH, and molecular results obtained after 1 year of follow-up. In our patient, the banding technique did not reveal the standard Ph, but
Fig. 2. FISH on a bone marrow metaphase cell using BCR-ABL dualcolor, dual-fusion probe. Arrow indicates the residual BCR probe signal on the derivative 22.
rather 2 apparently not specific numerical abnormalities, whereas D-FISH showed 1 or 2 BCR/ABL fusion signals, located on 9q34 instead of on 22q11, as usually occurs in standard Ph1 patients and in the rare Ph-negative, BCR/ ABL positive patients [8]. We can hypothesize that such a finding may be due to a second translocation involving the same chromosomes 9 and 22 that originally gave rise to the standard t(9;22), reconstituting the 2 chromosomes, which then appear cytogenetically normal [9]. Another explanation could be an insertion of BCR sequences (5’compared to the major breakpoint cluster region) into the ABL region (5’compared to exon 2). Such insertion could lead to the formation of the classical BCR/ ABL fusion gene that is responsible for the production of the p210 fusion protein. Moreover, FISH revealed that the cells with trisomy 9 showed a duplication of the chromosome 9 that carries the BCR/ABL fusion gene: this must be considered a Ph duplication. Similar observations were reported by Takahashi et al. [10]. It is usually assumed that the duplication of the Ph, which is frequently observed during disease progression, is due to non-disjunction, an event
Table 1 Cytogenetic, FISH, and molecular results at diagnosis and after 3, 6, and 12 months of GlivecÒ therapy Technique
Diagnosis
3 months
6 months
12 months
Banding
47,XY,121[15]/ 48,XY,19,121[16] 95%
46,XY [20]
46,XY [20]
46,XY [20]
8%
4%
4%
7,584
12.58
8.55
3.5
FISH [BCR/ABL fusion signal positive cells]a RT-Q-PCRb
The numbers in brackets refer to the number of observed metaphases. It is worth noting the differences in sensitivity in revealing the progressive reduction of the leukemic clone by banding, FISH, and RT-PCR. a Percentage of positive cells ! 5 is indicative of the absence of Ph. b Ratio ! 5 is considered borderline.
G. Fugazza et al. / Cancer Genetics and Cytogenetics 163 (2005) 173–175
that is still unclear [11]. Keeping in mind that trisomy 9 is a rare event in CML and in the advanced phases of disease as well, we can hypothesize that the duplication of this chromosome is favored by the presence of the BCR/ABL sequence on it. On the other hand, duplication of the derivative chromosome carrying the new gene that is responsible for the disease is not a rare event in leukemia [12–14]. D-FISH is usually performed to demonstrate the presence of a second fusion signal caused by the chimeric ABL/BCR gene on 9q34 [15], or if this signal is absent, to assess deletions of ABL sequences flanking the breakpoint region. This observation has acquired important prognostic significance [16]. In our patient with masked Ph, this prognostic value cannot be documented because the BCR/ABL signal is mapped exactly on 9q34. Lastly, we can state that therapy with Glivec proved to be efficient from a clinical, cytogenetic, and molecular point of view. In fact, RT-Q-PCR demonstrated that the quantitative measurement of the p210 fusion protein decreased from 7,584 before therapy to 12.58 after 3 months, to 8.54 after 6 months, and to 3.5 after 12 months of therapy with Glivec. In other words, despite the unusual cytogenetic features, therapy with Glivec (Novartis, Basel, Switzerland) led to complete and rapid hematological response and to complete cytogenetic response after 3 months with persisting BCR/ ABL positive cells, as revealed by the FISH and RT-QPCR techniques. After 6 months, FISH was negative, and the RT-Q-PCR became weakly positive. Lastly, after 1 year, the RT-Q-PCR value was !5 (borderline value). The unusual location of BCR/ABL sequences on a chromosome 9 and the duplication of the this chromosome apparently did not confer drug resistance.
[5]
[6]
[7]
[8]
[9]
[10]
[11] [12]
[13]
[14]
References [15] [1] Barnes DJ, Melo JV. Cytogenetic and molecular genetic aspects of chronic myeloid leukaemia. Acta Haematol 2002;108:180–202. [2] Bentz M, Cabot G, Moos M, Speicher MR, Ganser A, Lichter P, Dohner H. Detection of chimeric BCR-ABL genes on bone marrow samples and blood smears in chronic myeloid and acute lymphoblastic leukemia by in situ hybridization. Blood 1994;83:1922–8. [3] Wells SJ, Phillips CN, Winton EF, Farhi DC. Reverse transcriptasepolymerase chain reaction for bcr/abl fusion in chronic myelogenous leukemia. Am J Clin Pathol 1996;105:756–60. [4] Amabile M, Giannini B, Testoni N, Montefusco V, Rosti G, Zardini C, Terragna C, Buonamici S, Ottaviani E, Soverini S, Fiacchini M, Bassi S, de Vivo A, Trabacchi E, Saglio G, Pane F, Baccarani M, Tura S, Martinelli G. Real-time quantification of
[16]
175
different types of bcr-abl transcript in chronic myeloid leukemia. Haematologica 2001;86:252–9. Gabert J, Beillard E, van der Velden VH, Bi W, Grimwade D, Pallisgaard N, Barbany G, Cazzaniga G, Cayuela JM, Cave H, Pane F, Aerts JL, De Micheli D, Thirion X, Pradel V, Gonzalez M, Viehmann S, Malec M, Saglio G, van Dongen JJ. Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukaemia. A Europe Against Cancer Program. Leukemia 2003;17:2318–57. Kaeda J, Chase A, Goldman JM. Cytogenetic and molecular monitoring of residual disease in chronic myeloid leukaemia. Acta Haematol 2002;107:64–75. Fugazza G, Miglino M, Bruzzone R, Quintino S, Gatti AM, Grasso R, Gobbi M, Frassoni F, Sessarego M. Cytogenetic and fluorescence in situ hybridization monitoring in Ph1 chronic myeloid leukemia patients treated with imatinib mesylate. J Exp Clin Cancer Res 2004;23:295–9. Lazaridou A, Chase A, Melo J, Garicochea B, Diamond J, Goldman J. Lack of reciprocal translocation in BCR-ABL positive Ph-negative chronic myeloid leukemia. Leukemia 1994;8:454–7. Hagemeijer A, Buijs A, Smit E, Janssen B, Creemers GJ, Van der Plas D, Grosveld G. Translocation of BCR to chromosome 9: a new cytogenetic variant detected by FISH in two Ph-negative, BCR-positive patients with chronic myeloid leukemia. Genes Chromosomes Cancer 1993;8:237–45. Takahashi N, Miura I, Ohshima A, Utsumi S, Nimura T, Hashimoto K, Saito M, Miura AB. Duplication of chromosome 9 carrying a BCR/ABL chimeric gene in Philadelphia chromosome negative chronic myeloid leukemia. Cancer Genet Cytogenet 1996;89: 166–9. Lamb NE, Hassold TJ. Nondisjunction–a view from ringside. N Engl J Med 2004;351:1931–4. Auger MJ, Ross FM, Mackie MJ. 8;21 translocation with duplication of the der(21) in a patient with myelomonocytic leukemia. Cancer Genet Cytogenet 1991;51:139–41. Fugazza G, Cerri R, Bruzzone R, Patrone F, Sessarego M. Duplication of the der(13)t(12;13)(p13;q14) in chronic myelomonocytic leukemia. Haematologica 1997;82:336–7. Maserati E, Casali M, Invernizzi R, Brugnatelli S. Duplication of the der(14) chromosome of a translocation (8;14) in a case of Burkitt’s type L3-ALL. Cancer Genet Cytogenet 1992;61:218–9. Dewald G, Stallard R, Alsaadi A, Arnold S, Blough R, Ceperich TM, Rafael Elejalde B, Fink J, Higgins JV, Higgins RR, Hoeltge GA, Hsu WT, Johnson EB, Kronberger D, McCorquodale DJ, Meisner LF, Micale MA, Oseth L, Payne JS, Schwartz S, Sheldon S, Sophian A, Storto P, Van Tuinen P, Wenger GD, Wiktor A, Willis LA, Yung JF, Zenger-Hain J. A multicenter investigation with D-FISH BCR/ABL1 probes. Cancer Genet Cytogenet 2000;116:97–104. Huntly BJ, Reid AG, Bench AJ, Campbell LJ, Telford N, Shepherd P, Szer J, Prince HM, Turner P, Grace C, Nacheva EP, Green AR. Deletions of the derivative chromosome 9 occur at the time of the Philadelphia translocation and provide a powerful and independent prognostic indicator in chronic myeloid leukemia. Blood 2001;98: 1732–8.