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Identical abnormality of the short arm of chromosome 18 in two Philadelphia-positive chronic myelocytic leukemia patients with erythroblastic transformation, resulting in duplication of BCR-ABL1 fusion
Cancer Genetics and Cytogenetics 138 (2002) 22–26
Identical abnormality of the short arm of chromosome 18 in two Philadelphia-positive chronic myelocytic leukemia patients with erythroblastic transformation, resulting in duplication of BCR-ABL1 fusion Florence Nguyen Khaca,*, Marie-Christine Waillb, Serge P. Romanab, Isabelle Radford-Weissb, Maryvonne Bussonc, Marie-Agnès Collonge-Ramed, Antoine Ribadeau-Dumase, Marie-Claude Piffautf, Marie-Thérèse Danielg, Frédéric Davia, Hélène Merle-Bérala, Roland Bergerc, Michel Arocke a
Service d’Hématologie Biologique, Groupe Hospitalier Pitié-Salpêtrière, Paris, France b Service de Cytogénétique, Hôpital Necker-Enfants Malades, Paris, France c Inserm U434, IGM, Paris, France d Centre Hospitalier Universitaire de Besançon, Besançon, France e Laboratoire d’Hématologie Cellulaire et Moléculaire, UPRES EA 2509, Faculté de Pharmacie, Paris f Centre Hospitalier André Boulloche, Montbéliard, France g Laboratoire Central d’Hématologie, Hôpital Saint-Louis, Paris, France Received 17 December 2001; received in revised form 11 February 2002; accepted 12 February 2002
Abstract
Two patients with Ph-positive chronic myelocytic leukemia in erythroblastic transformation and rearrangement of the short arm of chromosome 18 are reported. Fluorescence in situ hybridization studies showed that the 18p rearrangement resulted from translocation of the main part of chromosome 22 long arm to 18p, including BCR-ABL1 fusion. The 18p abnormality resulted, thus, in loss of 18p and duplication of BCR-ABL1 in both patients. The possible relation to the erythroblastic type of blastic phase is briefly discussed. In addition an apparently intact germline ABL1 gene was duplicated and inserted into chromosome 6 at band p21 in one of these patients. © 2002 Elsevier Science Inc. All rights reserved.
1. Introduction The Philadelphia (Ph) chromosome is usually associated with the t(9;22)(q34;q11) and results in BCR-ABL1 fusion gene, which is the hallmark of chronic myelocytic leukemia (CML) (for review see [1]). CML evolution toward transformation to blast crisis is accompanied by additional chromosome abnormalities in more than 80% of CML cases. Most of the additional abnormalities appear to be nonrandom, and the most frequently observed are trisomy 8, isochromosome 17q, an extra Ph chromosome (often double Ph), and trisomy 19 [2,3]. Other uncommon abnormalities also occur in the blastic phase, in addition to t(9;22). Al-
though some associations between chromosome abnormalities and morphological and immunological features of blasts in acute myeloid transformation have been reported, the most obvious correlations remain those associated with t(15;17), 11q23 rearrangements and inv(16) in blast crisis of promyelocytic, monocytic, and myelomonocytic with eosinophilia types of transformation, respectively [4]. Here, we report two patients with Ph CML in blastic transformation with an important erythroblastic component, and sharing the same 18p abnormality. 2. Case reports 2.1. Patient 1
* Corresponding author. Service d’Hématologie Biologique, Groupe Hospitalier Pitié-Salpêtrière, Pavillon Laveran, 47–83 bd de l’Hôpital, 75013 Paris, France. Tel.: 33-1-4216-0216; fax: 33-1-4216-0210 E-mail address: [email protected] (F. Nguyen Khac).
An 87-year-old woman was admitted for an ischemic stroke in June 1999 to the Centre Hospitalier, Montbéliard, France. She had been previously treated for bladder cancer
0165-4608/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S0165-4608(02)00 5 7 4 - 5
F. Nguyen Khac et al. / Cancer Genetics and Cytogenetics 138 (2002) 22–26
by surgery alone. In the peripheral blood, the white blood cell count (WBC) was 20 109/L without immature blast cells; platelets were at 1.1 1012/L. The bone marrow was hypercellular, with numerous megakaryocytes. Physical examination found splenomegaly and there was no indication of evolution of the bladder cancer. The diagnosis was myeloproliferative disorder with a Ph chromosome. Treatment consisted of hydroxyurea, and then, of 6-mercaptopurine. In January 2001, she was admitted at the hospital for asthenia. Lymphnode and spleen enlargement was found at physical examination. In the peripheral blood, the WBC was 10.6 109/L with immature granulocytic cells (17%), blasts (1%) and erythroblasts (2%), 9.8 g/dL of hemoglobin, and 2.6 1012/L platelets. Treatment by pipobroman was started, but the patient died in May 2001, with a WBC of 25.6 109/L with 88% blasts, which were abnormal erythroblastic precursors (immunophenotype: CD36, CD71, glycophorin A, CD13, CD33, and CD7). 2.2. Patient 2 A 49-year-old woman was admitted for fatigue, weight loss, and fever to the Saint-Louis Hospital, Paris, France, in December 1984. She was known to have CML discovered in July 1981, because of abdominal pain related to considerable splenomegaly. At that time, hematological findings showed 46.2 109/L white cells in the peripheral blood with immature myeloid cells. Hemoglobin level and platelet count were normal, and bone marrow was hypercellular with granulocytic hyperplasia. She was treated with hydroxyurea, busulfan, and hydroxyurea-mercaptopurine-prednisone since March 1984. In December 1984, liver and spleen enlargement was found, and hematological findings were: in peripheral blood, 45.2 109/L white cells with 25% polymorphonuclear neutrophils cells, 16% eosinophils, 11% basophils, 9% metamyelocytes, 2% myelocytes, 2% promyelocytes, 16% blasts, and 14% monocytes, 9.2 g/dL hemoglobin, and 14 109/L platelets. In bone marrow, 4% hemoblasts, 66% granulocytic cells (3% myeloblasts, 8% promyelocytes, 3% myelocytes, 10% metamyelocytes, 21% polymorphonuclear neutrophils, 6% eosinophils and 15% basophils), 1% monocytes, 25% erythroblastic cells (5% proerythroblasts, 2% basophilic erythroblasts, 8% polychromatophils, and 10% acidophils), 4% plasmocytes, and presence of abnormal megakaryocytes (micromegakaryocytes and monolobulated megakaryocytes). The diagnosis was CML in accelerated phase with blastic component mainly composed by abnormal proerythroblasts. The patient was treated with bisanthrene, and left the hospital after two weeks with improved hematological findings without disappearance of abnormal blasts in the bone marrow. She died in January 1985, with a WBC of 80 109/L with 24% immature granulocytic cells and 17% blasts, which were abnormal erythroblastic precursors. 3. Materials and methods Chromosomes were prepared from bone marrow and peripheral blood cells after 24–48 h in vitro cultures in RPMI-
23
1640 medium (GIBCO BRL). Cytogenetic analysis was performed using RHG banding techniques as described [5], and chromosomes were classified according the International System for Human Cytogenetic Nomenclature (ISCN 1995) [6]. Fluorescence in situ hybridization (FISH) studies on metaphase chromosomes were performed in the acute phase in both patients with the usual techniques [7]. Several probes were used: whole chromosome 6, 8, 9, 18, and 22 painting probes (Vysis, Adgenix), and chromosome 18-specific alpha satellite probe (Appligene Oncor, Qbiogene). Two sets of probes were used to visualize the BCR and ABL1 rearrangements: LSI BCR/ABL ES dual-color translocation probe (LSI BCR green, LSI ASSABL red) (Vysis, Adgenix), as well as PAC dJ1132H12 and dJ835J22 (kindly provided by M. Rocci, University of Bari, Italy). The ASS-ABL probe spans both sides of the breakpoint on 9q so that Ph cells show colocalization of the BCR and ASSABL probes and a third independent fluorescent signal corresponding to ASS-ABL probe (split signal remaining on the derivative chromosome 9). The BCR probe does not give any signal on the derivative chromosome 9 if the breakpoint is located within the M-BCR region. PAC dJ1132H12 is covering the 5 part of ABL1 beginning in the first intron, and PAC dJ835J22 is covering the 3 part of ABL1 beginning in exon 11. For spectral karyotype analysis, probes used, hybridization, and image acquisition were performed as previously described [8]. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed using standard techniques [9]. 4. Results 4.1. Cytogenetics 4.1.1. Patient 1 At diagnosis in June 1999, chromosome studies, performed on bone marrow cells, showed only abnormal karyotypes: 46,XX,t(9;22)(q34;q11)[18]/47,idem,8[2]. Nineteen months later, at the beginning of the blast crisis in January 2001, chromosome studies were performed on unstimulated peripheral blood cells. The 20 metaphase cells examined showed the karyotype, 47,XX,8,t(9;22)(q34;q11),add(18) (p11) (Fig. 1a). 4.1.2. Patient 2 At the beginning of the blast crisis in December 1984, R-banded chromosome analysis on bone marrow cells performed after 48 hours-in vitro culture showed the abnormal karyotype 47,XX,t(9;22)(q34;q11),add(18)(p11),19, in 20 cells (Fig. 1b). 4.2. FISH We first used the LSI BCR/ABL extra signal (ES) dualcolor translocation probe. As a result, dual-color FISH metaphase chromosomes confirmed the presence of the t(9;22) in the two patients, without remaining signal on the derivative chromosome 9. The two probes BCR and ASS-ABL were also colocalized on one 18p in both patients. The use of chromosome-specific painting probes confirmed these findings. Furthermore, loss of material of the short arm of
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F. Nguyen Khac et al. / Cancer Genetics and Cytogenetics 138 (2002) 22–26
Fig. 1. R-banded partial karyotypes of patients 1 (a) and 2 (b). Arrowheads indicate the add(18)(p11).
the derivative chromosome 18 was observed with the whole chromosome 18 painting probe in patient 1. The use of chromosome 18 -satellite probe confirmed the rearrangement of 18p in the two patients. In addition, a red signal corresponding to the ASS-ABL probe was present on the short arm of one chromosome 6 at band p21, in metaphases of patient 1 (Fig. 2a and b). In this patient, hybridization with PAC dJ835J22 (3 part of ABL1) showed the presence of signals on the normal 9q, der(22), 18p, and 6p. Hybridization with PAC dJ1132H12 (5 part of ABL1) showed a signal on the normal 9q and 6p. In patient 2, the use of PAC dJ835J22 showed signals on the normal 9q, der(22), and 18p. Only one signal was present on the normal 9q with probe dJ1132H12, confirming loss of 5 part of ABL1 in the der(9). To detect another potential cryptic rearrangement, spectral karyotyping was performed on five metaphase cells of case 1. No additional chromosome abnormality was observed, and we noted that the insertion of chromosome 9 into the short arm of chromosome 6 was not detected with this technique (Fig. 2c). In conclusion, FISH analysis was in accordance with the localization of chromosome 22 breakpoints within M-BCR and showed that BCR-ABL1 fusion was duplicated, with signals on 18p in both patients. In addition, the 5 part of ABL1 was lost in the rearranged 9q in both patients, and a fragment of 9q containing at least a large part of ABL1 was inserted in 6p21 in patient 1. Chromosome 18 rearrangement also resulted in deletion, at least partial, of the short arm. 4.3. RT-PCR RT-PCR analysis confirmed the BCR-ABL1 fusion with b3a2 junction, which indicated the breakpoint within the major breakpoint cluster region (M-BCR) in patient 1. Shortage of material precluded RT-PCR analysis in patient 2.
5. Discussion Duplication of the Ph chromosome resulting in duplication of the chimeric BCR-ABL1 gene is frequently observed in various cytological types of CML blast crisis. An extra copy of the Ph chromosome is also observed in some patients as a dicentric Ph chromosome or as an isochromosome derivative 22q. Finally, uncommon cases of transposition of duplicated BCR-ABL1 have been reported on chromosomes 17 [10], 12, and 18 [11]. The 18p abnormality of the present cases is equivalent to a Ph duplication and was proven to have appeared secondarily in patient 1. The use of the BCR/ABL ES probe allows identification 5-ABL1 deletion associated with loss of the arginosuccinate synthetase gene (ASS). Deletion of the 5-ABL1 region is a recurrent abnormality detected in about 15% of Ph-positive CML patients and has been claimed to have a prognostic significance [12–14]. A 5-ABL1 deletion was observed by FISH analysis in patient 2. In patient 1, a signal corresponding to the ABL1 locus was unexpectedly detected on the short arm of the chromosome 6, although this was not visible with spectral karyotyping, probably because of the small size of the inserted fragment. Insertion of complete or at least a large part of ABL1 within 6p indicates that ABL1 duplication occurred prior to its translocation to chromosome 6. This rearrangement followed at least three chromosomal breakpoints (two on 9q, 5 and 3 to ABL1, and one on 6p). Chronic phase samples were not available for FISH analysis. Although the mechanism of this rearrangement is not established, complete or partial ABL1 duplication has also been reported by others [11,15]. Erythroblastic transformation of CML has been reported in 10% of patients in blastic crisis, and no specific aberration has been described for this type of transformation [2,3]. Trisomy 8 (as in patient 1) and trisomy 19 (as in patient 2)
F. Nguyen Khac et al. / Cancer Genetics and Cytogenetics 138 (2002) 22–26
25
Fig. 2. FISH analysis with LSI BCR/ABL-ES dual-color probe (BCR appears in green and ABL in red). (a) Patient 1. (b) Patient 2. (c) Spectral karyotype of one metaphase spread of patient 1 after spectra-based classification (pseudocolor). Note chromosomes 6 appear normal.
are frequently observed in blast crisis of CML and are not known to be specifically associated with the erythroblastic transformation type. Duplication and transposition of BCRABL1 were present in the two patients reported here. The rearrangement of 18p occurred after the common t(9;22) in the first patient. The same sequence of events might occur in the second patient although simultaneous occurrence of these events cannot be excluded. The presence of the translocations in two patients with CML with erythroblastic transformation makes it possible that the translocation of
the fused BCR-ABL1 gene on the short arm of chromosome 18 may be associated with this particular cytological subtype of disease progression in CML. To our knowledge, only one case of accelerated phase of CML with a transposition of duplicated chromosomal segment involving fused BCR-ABL1 gene on the short arm of the chromosome 18 has been reported in the literature [11]. The blast morphology, however, was not detailed. Furthermore, loss of the major part of the short arm of chromosome 18 could also be a significant event for disease progression.
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F. Nguyen Khac et al. / Cancer Genetics and Cytogenetics 138 (2002) 22–26
Acknowledgments We thank Gaëlle Le Guyader, Sylvie Nusbaum, and Marylène Belluteau for expert technical assistance.
[10]
References [1] Deininger MW, Goldman JM, Melo JV. The molecular biology of chronic myeloid leukemia. Blood 2000;96:3343–56. [2] Mitelman F, Johansson B, Mertens F. Mitelman database of chromosome aberrations in cancer. http://cgap.nci.nih.gov/Chromosomes/ Mitelman, 2001. [3] Sandberg AA. The chromosomes in human cancer and leukemia 2nd edition. New York: Elsevier, 1990. [4] Heim S, Mitelman F. New York: Wiley, 1995. [5] Dutrillaux B, Lejeune J. A new technic of analysis of the human karyotype. C R Acad Sci Hebd Seances Acad Sci D 1971;272:2638–40. [6] ISCN. An international system for human cytogenetic nomenclature. Mitelman F, editor. Basel: S. Karger, 1995. [7] Le Coniat M, Romana SP, Berger R. Partial chromosome 21 amplification in a child with acute lymphoblastic leukemia. Genes Chromosomes Cancer 1995;14:204–9. [8] Elghezal H, Le Guyader G, Radford-Weiss I, Perot C, Van Den Akker J, Eydoux P, Vekemans M, Romana SP. Reassessment of childhood B-lineage lymphoblastic leukemia karyotypes using spectral analysis. Genes Chromosomes Cancer 2001;30:383–92. [9] van Dongen JJ, Macintyre EA, Gabert JA, Delabesse E, Rossi V, Saglio G, Gottardi E, Rambaldi A, Dotti G, Griesinger F, Parreira A, Gameiro P, Diaz MG, Malec M, Langerak AW, San Miguel JF, Biondi A. Standardized RT-PCR analysis of fusion gene transcripts from chromosome
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aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia 1999;13:1901–28. Metzke-Heidemann S, Harder L, Gesk S, Schoch R, Jenisch S, Grote W, Siebert R, Schlegelberger B. Integration of amplified BCR/ABL fusion genes into the short arm of chromosome 17 as a novel mechanism of disease progression in chronic myeloid leukemia. Genes Chromosomes Cancer 2001;31:10–4. Tanaka K, Arif M, Kyo T, Dohy H, Kamada N. Transposition of duplicated chromosomal segment involving fused BCR-ABL gene or ABL oncogene alone in chronic myelocytic leukemia and Ph chromosome-positive acute leukemia with complex karyotypes. Cancer Genet Cytogenet 2000;119:8–14. Dewald GW, Wyatt WA, Juneau AL, Carlson RO, Zinsmeister AR, Jalal SM, Spurbeck JL, Silver RT. Highly sensitive fluorescence in situ hybridization method to detect double BCR/ABL fusion and monitor response to therapy in chronic myeloid leukemia. Blood 1998;91:3357–65. Herens C, Tassin F, Lemaire V, Beguin Y, Collard E, Lampertz S, Croisiau C, Lecomte M, De Prijk B, Longree L, Koulischer L. Deletion of the 5-ABL region: a recurrent anomaly detected by fluorescence in situ hybridization in about 10% of Philadelphia-positive chronic myeloid leukaemia patients. Br J Haematol 2000;110:214–6. Sinclair PB, Nacheva EP, Leversha M, Telford N, Chang J, Reid A, Bench A, Champion K, Huntly B, Green AR. Large deletions at the t(9; 22) breakpoint are common and may identify a poor-prognosis subgroup of patients with chronic myeloid leukemia. Blood 2000;95:738–43. Tanaka K, Arif M, Eguchi M, Kyo T, Dohy H, Kamada N. Frequent jumping translocations of chromosomal segments involving the ABL oncogene alone or in combination with CD3-MLL genes in secondary leukemias. Blood 1997;89:596–600.
Identical abnormality of the short arm of chromosome 18 in two Philadelphia-positive chronic myelocytic leukemia patients with erythroblastic transformation, resulting in duplication of BCR-ABL1 fusion Florence Nguyen Khaca,*, Marie-Christine Waillb, Serge P. Romanab, Isabelle Radford-Weissb, Maryvonne Bussonc, Marie-Agnès Collonge-Ramed, Antoine Ribadeau-Dumase, Marie-Claude Piffautf, Marie-Thérèse Danielg, Frédéric Davia, Hélène Merle-Bérala, Roland Bergerc, Michel Arocke a
Service d’Hématologie Biologique, Groupe Hospitalier Pitié-Salpêtrière, Paris, France b Service de Cytogénétique, Hôpital Necker-Enfants Malades, Paris, France c Inserm U434, IGM, Paris, France d Centre Hospitalier Universitaire de Besançon, Besançon, France e Laboratoire d’Hématologie Cellulaire et Moléculaire, UPRES EA 2509, Faculté de Pharmacie, Paris f Centre Hospitalier André Boulloche, Montbéliard, France g Laboratoire Central d’Hématologie, Hôpital Saint-Louis, Paris, France Received 17 December 2001; received in revised form 11 February 2002; accepted 12 February 2002
Abstract
Two patients with Ph-positive chronic myelocytic leukemia in erythroblastic transformation and rearrangement of the short arm of chromosome 18 are reported. Fluorescence in situ hybridization studies showed that the 18p rearrangement resulted from translocation of the main part of chromosome 22 long arm to 18p, including BCR-ABL1 fusion. The 18p abnormality resulted, thus, in loss of 18p and duplication of BCR-ABL1 in both patients. The possible relation to the erythroblastic type of blastic phase is briefly discussed. In addition an apparently intact germline ABL1 gene was duplicated and inserted into chromosome 6 at band p21 in one of these patients. © 2002 Elsevier Science Inc. All rights reserved.
1. Introduction The Philadelphia (Ph) chromosome is usually associated with the t(9;22)(q34;q11) and results in BCR-ABL1 fusion gene, which is the hallmark of chronic myelocytic leukemia (CML) (for review see [1]). CML evolution toward transformation to blast crisis is accompanied by additional chromosome abnormalities in more than 80% of CML cases. Most of the additional abnormalities appear to be nonrandom, and the most frequently observed are trisomy 8, isochromosome 17q, an extra Ph chromosome (often double Ph), and trisomy 19 [2,3]. Other uncommon abnormalities also occur in the blastic phase, in addition to t(9;22). Al-
though some associations between chromosome abnormalities and morphological and immunological features of blasts in acute myeloid transformation have been reported, the most obvious correlations remain those associated with t(15;17), 11q23 rearrangements and inv(16) in blast crisis of promyelocytic, monocytic, and myelomonocytic with eosinophilia types of transformation, respectively [4]. Here, we report two patients with Ph CML in blastic transformation with an important erythroblastic component, and sharing the same 18p abnormality. 2. Case reports 2.1. Patient 1
* Corresponding author. Service d’Hématologie Biologique, Groupe Hospitalier Pitié-Salpêtrière, Pavillon Laveran, 47–83 bd de l’Hôpital, 75013 Paris, France. Tel.: 33-1-4216-0216; fax: 33-1-4216-0210 E-mail address: [email protected] (F. Nguyen Khac).
An 87-year-old woman was admitted for an ischemic stroke in June 1999 to the Centre Hospitalier, Montbéliard, France. She had been previously treated for bladder cancer
0165-4608/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S0165-4608(02)00 5 7 4 - 5
F. Nguyen Khac et al. / Cancer Genetics and Cytogenetics 138 (2002) 22–26
by surgery alone. In the peripheral blood, the white blood cell count (WBC) was 20 109/L without immature blast cells; platelets were at 1.1 1012/L. The bone marrow was hypercellular, with numerous megakaryocytes. Physical examination found splenomegaly and there was no indication of evolution of the bladder cancer. The diagnosis was myeloproliferative disorder with a Ph chromosome. Treatment consisted of hydroxyurea, and then, of 6-mercaptopurine. In January 2001, she was admitted at the hospital for asthenia. Lymphnode and spleen enlargement was found at physical examination. In the peripheral blood, the WBC was 10.6 109/L with immature granulocytic cells (17%), blasts (1%) and erythroblasts (2%), 9.8 g/dL of hemoglobin, and 2.6 1012/L platelets. Treatment by pipobroman was started, but the patient died in May 2001, with a WBC of 25.6 109/L with 88% blasts, which were abnormal erythroblastic precursors (immunophenotype: CD36, CD71, glycophorin A, CD13, CD33, and CD7). 2.2. Patient 2 A 49-year-old woman was admitted for fatigue, weight loss, and fever to the Saint-Louis Hospital, Paris, France, in December 1984. She was known to have CML discovered in July 1981, because of abdominal pain related to considerable splenomegaly. At that time, hematological findings showed 46.2 109/L white cells in the peripheral blood with immature myeloid cells. Hemoglobin level and platelet count were normal, and bone marrow was hypercellular with granulocytic hyperplasia. She was treated with hydroxyurea, busulfan, and hydroxyurea-mercaptopurine-prednisone since March 1984. In December 1984, liver and spleen enlargement was found, and hematological findings were: in peripheral blood, 45.2 109/L white cells with 25% polymorphonuclear neutrophils cells, 16% eosinophils, 11% basophils, 9% metamyelocytes, 2% myelocytes, 2% promyelocytes, 16% blasts, and 14% monocytes, 9.2 g/dL hemoglobin, and 14 109/L platelets. In bone marrow, 4% hemoblasts, 66% granulocytic cells (3% myeloblasts, 8% promyelocytes, 3% myelocytes, 10% metamyelocytes, 21% polymorphonuclear neutrophils, 6% eosinophils and 15% basophils), 1% monocytes, 25% erythroblastic cells (5% proerythroblasts, 2% basophilic erythroblasts, 8% polychromatophils, and 10% acidophils), 4% plasmocytes, and presence of abnormal megakaryocytes (micromegakaryocytes and monolobulated megakaryocytes). The diagnosis was CML in accelerated phase with blastic component mainly composed by abnormal proerythroblasts. The patient was treated with bisanthrene, and left the hospital after two weeks with improved hematological findings without disappearance of abnormal blasts in the bone marrow. She died in January 1985, with a WBC of 80 109/L with 24% immature granulocytic cells and 17% blasts, which were abnormal erythroblastic precursors. 3. Materials and methods Chromosomes were prepared from bone marrow and peripheral blood cells after 24–48 h in vitro cultures in RPMI-
23
1640 medium (GIBCO BRL). Cytogenetic analysis was performed using RHG banding techniques as described [5], and chromosomes were classified according the International System for Human Cytogenetic Nomenclature (ISCN 1995) [6]. Fluorescence in situ hybridization (FISH) studies on metaphase chromosomes were performed in the acute phase in both patients with the usual techniques [7]. Several probes were used: whole chromosome 6, 8, 9, 18, and 22 painting probes (Vysis, Adgenix), and chromosome 18-specific alpha satellite probe (Appligene Oncor, Qbiogene). Two sets of probes were used to visualize the BCR and ABL1 rearrangements: LSI BCR/ABL ES dual-color translocation probe (LSI BCR green, LSI ASSABL red) (Vysis, Adgenix), as well as PAC dJ1132H12 and dJ835J22 (kindly provided by M. Rocci, University of Bari, Italy). The ASS-ABL probe spans both sides of the breakpoint on 9q so that Ph cells show colocalization of the BCR and ASSABL probes and a third independent fluorescent signal corresponding to ASS-ABL probe (split signal remaining on the derivative chromosome 9). The BCR probe does not give any signal on the derivative chromosome 9 if the breakpoint is located within the M-BCR region. PAC dJ1132H12 is covering the 5 part of ABL1 beginning in the first intron, and PAC dJ835J22 is covering the 3 part of ABL1 beginning in exon 11. For spectral karyotype analysis, probes used, hybridization, and image acquisition were performed as previously described [8]. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed using standard techniques [9]. 4. Results 4.1. Cytogenetics 4.1.1. Patient 1 At diagnosis in June 1999, chromosome studies, performed on bone marrow cells, showed only abnormal karyotypes: 46,XX,t(9;22)(q34;q11)[18]/47,idem,8[2]. Nineteen months later, at the beginning of the blast crisis in January 2001, chromosome studies were performed on unstimulated peripheral blood cells. The 20 metaphase cells examined showed the karyotype, 47,XX,8,t(9;22)(q34;q11),add(18) (p11) (Fig. 1a). 4.1.2. Patient 2 At the beginning of the blast crisis in December 1984, R-banded chromosome analysis on bone marrow cells performed after 48 hours-in vitro culture showed the abnormal karyotype 47,XX,t(9;22)(q34;q11),add(18)(p11),19, in 20 cells (Fig. 1b). 4.2. FISH We first used the LSI BCR/ABL extra signal (ES) dualcolor translocation probe. As a result, dual-color FISH metaphase chromosomes confirmed the presence of the t(9;22) in the two patients, without remaining signal on the derivative chromosome 9. The two probes BCR and ASS-ABL were also colocalized on one 18p in both patients. The use of chromosome-specific painting probes confirmed these findings. Furthermore, loss of material of the short arm of
24
F. Nguyen Khac et al. / Cancer Genetics and Cytogenetics 138 (2002) 22–26
Fig. 1. R-banded partial karyotypes of patients 1 (a) and 2 (b). Arrowheads indicate the add(18)(p11).
the derivative chromosome 18 was observed with the whole chromosome 18 painting probe in patient 1. The use of chromosome 18 -satellite probe confirmed the rearrangement of 18p in the two patients. In addition, a red signal corresponding to the ASS-ABL probe was present on the short arm of one chromosome 6 at band p21, in metaphases of patient 1 (Fig. 2a and b). In this patient, hybridization with PAC dJ835J22 (3 part of ABL1) showed the presence of signals on the normal 9q, der(22), 18p, and 6p. Hybridization with PAC dJ1132H12 (5 part of ABL1) showed a signal on the normal 9q and 6p. In patient 2, the use of PAC dJ835J22 showed signals on the normal 9q, der(22), and 18p. Only one signal was present on the normal 9q with probe dJ1132H12, confirming loss of 5 part of ABL1 in the der(9). To detect another potential cryptic rearrangement, spectral karyotyping was performed on five metaphase cells of case 1. No additional chromosome abnormality was observed, and we noted that the insertion of chromosome 9 into the short arm of chromosome 6 was not detected with this technique (Fig. 2c). In conclusion, FISH analysis was in accordance with the localization of chromosome 22 breakpoints within M-BCR and showed that BCR-ABL1 fusion was duplicated, with signals on 18p in both patients. In addition, the 5 part of ABL1 was lost in the rearranged 9q in both patients, and a fragment of 9q containing at least a large part of ABL1 was inserted in 6p21 in patient 1. Chromosome 18 rearrangement also resulted in deletion, at least partial, of the short arm. 4.3. RT-PCR RT-PCR analysis confirmed the BCR-ABL1 fusion with b3a2 junction, which indicated the breakpoint within the major breakpoint cluster region (M-BCR) in patient 1. Shortage of material precluded RT-PCR analysis in patient 2.
5. Discussion Duplication of the Ph chromosome resulting in duplication of the chimeric BCR-ABL1 gene is frequently observed in various cytological types of CML blast crisis. An extra copy of the Ph chromosome is also observed in some patients as a dicentric Ph chromosome or as an isochromosome derivative 22q. Finally, uncommon cases of transposition of duplicated BCR-ABL1 have been reported on chromosomes 17 [10], 12, and 18 [11]. The 18p abnormality of the present cases is equivalent to a Ph duplication and was proven to have appeared secondarily in patient 1. The use of the BCR/ABL ES probe allows identification 5-ABL1 deletion associated with loss of the arginosuccinate synthetase gene (ASS). Deletion of the 5-ABL1 region is a recurrent abnormality detected in about 15% of Ph-positive CML patients and has been claimed to have a prognostic significance [12–14]. A 5-ABL1 deletion was observed by FISH analysis in patient 2. In patient 1, a signal corresponding to the ABL1 locus was unexpectedly detected on the short arm of the chromosome 6, although this was not visible with spectral karyotyping, probably because of the small size of the inserted fragment. Insertion of complete or at least a large part of ABL1 within 6p indicates that ABL1 duplication occurred prior to its translocation to chromosome 6. This rearrangement followed at least three chromosomal breakpoints (two on 9q, 5 and 3 to ABL1, and one on 6p). Chronic phase samples were not available for FISH analysis. Although the mechanism of this rearrangement is not established, complete or partial ABL1 duplication has also been reported by others [11,15]. Erythroblastic transformation of CML has been reported in 10% of patients in blastic crisis, and no specific aberration has been described for this type of transformation [2,3]. Trisomy 8 (as in patient 1) and trisomy 19 (as in patient 2)
F. Nguyen Khac et al. / Cancer Genetics and Cytogenetics 138 (2002) 22–26
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Fig. 2. FISH analysis with LSI BCR/ABL-ES dual-color probe (BCR appears in green and ABL in red). (a) Patient 1. (b) Patient 2. (c) Spectral karyotype of one metaphase spread of patient 1 after spectra-based classification (pseudocolor). Note chromosomes 6 appear normal.
are frequently observed in blast crisis of CML and are not known to be specifically associated with the erythroblastic transformation type. Duplication and transposition of BCRABL1 were present in the two patients reported here. The rearrangement of 18p occurred after the common t(9;22) in the first patient. The same sequence of events might occur in the second patient although simultaneous occurrence of these events cannot be excluded. The presence of the translocations in two patients with CML with erythroblastic transformation makes it possible that the translocation of
the fused BCR-ABL1 gene on the short arm of chromosome 18 may be associated with this particular cytological subtype of disease progression in CML. To our knowledge, only one case of accelerated phase of CML with a transposition of duplicated chromosomal segment involving fused BCR-ABL1 gene on the short arm of the chromosome 18 has been reported in the literature [11]. The blast morphology, however, was not detailed. Furthermore, loss of the major part of the short arm of chromosome 18 could also be a significant event for disease progression.
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F. Nguyen Khac et al. / Cancer Genetics and Cytogenetics 138 (2002) 22–26
Acknowledgments We thank Gaëlle Le Guyader, Sylvie Nusbaum, and Marylène Belluteau for expert technical assistance.
[10]
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