- Email: [email protected]
RARA Chimeric Gene on Chromosome 2 in a Patient with Acute Promyelocytic Leukemia (M3) Associated with a New Variant Translocation
SHORT COMMUNICATION
A PML/RARA Chimeric Gene on Chromosome 2 in a Patient with Acute Promyelocytic Leukemia (M3) Associated with a New Variant Translocation: t(2;15;17) (q21;q22;q21) Masumi Fujishima, Naoto Takahashi, Ikuo Miura, Yoshimi Kobayashi, Masaaki Kume, Tamio Nishinari, and Akira B. Miura
ABSTRACT: We describe a patient with acute promyelocytic leukemia (APL) carrying a new complex variant translocation of t(2;15;17)(q21;q22;q21). The karyotypic interpretation was confirmed by fluorescence in situ hybridization (FISH) with the use of painting probes of chromosomes 2, 15, and 17 and a PML/RARA dual color DNA probe. FISH showed a PML/RARA fusion gene on the der(2) instead of the der(15). These results suggest that the critical event in the development of APL is the formation of a PML/RARA chimeric gene, regardless of its locus in the genome. © 2000 Elsevier Science Inc. All rights reserved.
INTRODUCTION The translocation t(15;17) is a special chromosome abnormality exclusively associated with acute promyelocytic leukemia (APL; M3 subtype according to French–American–British (FAB) classification) [1]. Although more than 90% of APL patients have this translocation, only a few patients have variant translocations [2, 3]. The case presented here showed a complex variant translocation, t(2;15;17)(q21;q22;q21) and a PML/RARA fusion gene on the der(2) instead of the der(15). MATERIAL AND METHODS Case Report A 77-year-old man visited Yuri General Hospital because of purpura. He had been well until August 1998, when he started to feel general fatigue. On admission, he showed a high-grade fever, purpura in the upper and lower extremities, and a conjunctival hemorrhage of the left eye. Organomegaly From the Third Department of Internal Medicine (M. F., N. T., I. M., M. K., A. B. M.) and Department of Clinical Laboratories (Y. K.), Akita University School of Medicine, and the Department of Internal Medicine, Yuri General Hospital (T. N.), Akita, Japan. Address reprint requests to: Ikuo Miura, M.D., Ph.D., Third Department of Internal Medicine, Akita University School of Medicine, 1-1-1, Hondo, Akita 010-8543, Japan. Received August 16, 1999; accepted November 5, 1999. Cancer Genet Cytogenet 120:80–82 (2000) 2000 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
was not observed. The hemoglobin level was 9.7 g/dL and the white blood cell count was 0.7 ⫻ 109/L with 27% of leukemic promyelocytes. The platelet count was 16 ⫻ 109/L. Coagulation tests revealed a prothrombin time (PT) of 12.7 sec (10.9 ⵑ 12.5 sec), fibrinogen level of 534 mg/dL and fibrin degradation products (FDP) of more than 160 mg/ml. The bone marrow was hyperplastic with 85% atypical promyelocytes, which were packed with numerous azurophilic granules and Auer rods. Leukemic cells were CD13⫹, CD33⫹, CD34⫺, and HLA-DR⫺. A diagnosis of APL was made and all-trans retinoic acid was administered at a daily dose of 60 mg. A complete remission was achieved and chemotherapy including anthracyclines was followed with no recurrence to date. Cytogenetics Chromosome analysis was carried out according to a standard procedure [3]. G-banded chromosomes were designated and described according to ISCN 1995 [4]. Fluorescence In Situ Hybridization Cells kept in fixative at ⫺20⬚C were used for fluorescence in situ hybridization (FISH) analysis. Painting probes of chromosomes 2 and 15 labeled with biotin (Cambio, Cambridge) and of chromosome 17 with digoxigenin (Oncor, Gaithersburg, MD) and a PML/RARA dual-color DNA probe (Vysis, Fujisawa Co. Ltd., Osaka) were used. After hybridization, detection was performed with fluoresceinlabeled avidin/rhodamine-labeled anti-digoxigenin (On-
0165-4608/00/$–see front matter PII S0165-4608(99)00238-1
PML/RARA on 2q in APL with t(2;15;17)
Figure 1 G-banded karyotype of the bone marrow: 45,X,⫺Y,t(2;15;17)(q21;q22;q21). Figure 2 FISH study with painting probes of chromosomes 2 (green), 15 (green), and 17 (red), and with probes of the PML gene (red) and the RARA gene (green). (A) The green signals show chromosome 15 with FITC staining and the red signals show chromosome 17 with rhodamine staining. (B) The green signals show chromosome 2 with FITC staining and the red signals show chromosome 17 with rhodamine staining. (C) Probes of the PML gene (red) appear yellow on chromosome 15 with FITC staining, and PML-RARA fusion signal is seen on a chromosome other than chromosome 15. (D) Chromosome 2 is labeled with FITC (green) and the PML gene (red) is on chromosome 15. PML-RARA fusion signal is seen on the der(2).
81
82
M. Fujishima et al.
cor). The chromosomes were identified by 4⬘-6-diamidino2-phenylindole (DAPI) stain. Fluorescent signals were visualized under a Nikon microscope (Tokyo, Japan) with UV-2A and FITC/rhodamine dual band filters (Nikon). RESULTS Chromosome Analysis The chromosomal aberration was interpreted as 46,XY, t(2;15;17)(q21;q22;q21) [20] (Fig. 1). FISH Analysis The FISH results are illustrated in Figure 2. The chromosome 15 probe (green) hybridized to one chromosome 17 and the chromosome 17 probe (red) hybridized to the other chromosome (Fig. 2A). The chromosome 17 probe (red) hybridized to one chromosome 2 and the chromosome 2 probe (green) hybridized to another chromosome (Fig. 2B). FISH analysis with painting probes supported the karyotypic interpretation of the t(2;15;17). Red signals of the PML gene are on chromosome 15, while the PML/ RARA fusion signal was located on a long chromosome other than chromosome 15 or 17 (Fig. 2C). The PML/ RARA fusion signal was seen on the der(2) (Fig. 2D). These FISH analyses reveal 46,XY,t(2;15;17)(q21;q22;q21) .ish t(2;15;17)(wcp2⫹,PML⫹,RARA⫹,wcp17⫹; wcp15⫹, PML⫺,wcp2⫹; wcp17⫹,RARA⫺,wcp15⫹). DISCUSSION The reciprocal translocation t(15;17) is specifically associated with APL [1] and results in a PML-RARA fusion gene on the der(15). We described an APL patient with t(2;15;17), a complex variant translocation of t(15;17), that was supported by FISH. There have been several complex variant translocations reported in the literature, including t(2;17;15) (q21;q21;q22–24) [2] and t(15;19;17)(q22;p13;q12) [3]. Although the break points of t(2;17;15)(q21;q21;q22–24) [2] were shown to be the same as in our patient, the t(2;17;15) (q21;q21;q22–24) was observed in a second line and was masked by the part of chromosome 2q. We have reported a duplication of BCR-ABL fusion gene on chromosome 9 instead of der(22) in a patient with Ph negative BCR positive CML [5]. Similar events were found in this patient. FISH analysis of our patient showed the fusion PML-RARA gene on the der(2) instead of the der(15). Although the actual order of events could not be confirmed, one hypothetical scheme is shown in Figure 3 namely, to that a standard t(15;17) occurred followed by a translocation between chromosome 2 and the der(15). Since we did not observe a cell with only the t(15;17) in this study, the
Figure 3 Schematic presentation of t(2;15;17) by stepwise translocation. A standard t(15;17) is formed at the first step. As a second step, a translocation between the der(15) and chromosome 2 occurs, PML/RARA fusion gene moves to chromosome 2. possibility that all the events happened at the same time and not in two steps cannot be ruled out. The critical event of APL is the formation of the PML-RARA chimeric gene, regardless of its location in the genome.
REFERENCES 1. Rowley JD, Golomb HM, Dougherty C (1977): 15/17 translocation, a consistent chromosomal change in acute promyelocytic leukaemia. Lancet 1:549–550. 2. Bjerrum OW, Philip P, Pressler T, Tygstrup I (1987): Acute promyelocytic leukemia with t(15;17) and t(2;17;15). Cancer Genet Cytogenet 28:107–111. 3. Saitoh K, Miura I, Kobayashi Y, Kume M, Utsumi S, Takahashi N, Hatano Y, Nimura T, Hashimoto K, Takahashi S, Miura AB (1998): A new variant translocation of t(15;17) in a patient with acute promyelocytic leukemia (M3): t(15;19;17) (q22;p13;q12). Cancer Genet Cytogenet 102:15–18. 4. ISCN (1995): An International System for Human Cytogenetic Nomenclature. F Mitelman, ed. S. Karger, Basel. 5. Takahashi N, Miura I, Ohshima A, Utsumi S, Nimura T, Hashimoto K, Saitoh M, Miura AB (1996): Duplication of chromosome 9 carrying a BCR/ABL chimeric gene in Philadelphia chromosome negative chronic myeloid leukemia. Cancer Genet Cytogenet 89:166–169.
A PML/RARA Chimeric Gene on Chromosome 2 in a Patient with Acute Promyelocytic Leukemia (M3) Associated with a New Variant Translocation: t(2;15;17) (q21;q22;q21) Masumi Fujishima, Naoto Takahashi, Ikuo Miura, Yoshimi Kobayashi, Masaaki Kume, Tamio Nishinari, and Akira B. Miura
ABSTRACT: We describe a patient with acute promyelocytic leukemia (APL) carrying a new complex variant translocation of t(2;15;17)(q21;q22;q21). The karyotypic interpretation was confirmed by fluorescence in situ hybridization (FISH) with the use of painting probes of chromosomes 2, 15, and 17 and a PML/RARA dual color DNA probe. FISH showed a PML/RARA fusion gene on the der(2) instead of the der(15). These results suggest that the critical event in the development of APL is the formation of a PML/RARA chimeric gene, regardless of its locus in the genome. © 2000 Elsevier Science Inc. All rights reserved.
INTRODUCTION The translocation t(15;17) is a special chromosome abnormality exclusively associated with acute promyelocytic leukemia (APL; M3 subtype according to French–American–British (FAB) classification) [1]. Although more than 90% of APL patients have this translocation, only a few patients have variant translocations [2, 3]. The case presented here showed a complex variant translocation, t(2;15;17)(q21;q22;q21) and a PML/RARA fusion gene on the der(2) instead of the der(15). MATERIAL AND METHODS Case Report A 77-year-old man visited Yuri General Hospital because of purpura. He had been well until August 1998, when he started to feel general fatigue. On admission, he showed a high-grade fever, purpura in the upper and lower extremities, and a conjunctival hemorrhage of the left eye. Organomegaly From the Third Department of Internal Medicine (M. F., N. T., I. M., M. K., A. B. M.) and Department of Clinical Laboratories (Y. K.), Akita University School of Medicine, and the Department of Internal Medicine, Yuri General Hospital (T. N.), Akita, Japan. Address reprint requests to: Ikuo Miura, M.D., Ph.D., Third Department of Internal Medicine, Akita University School of Medicine, 1-1-1, Hondo, Akita 010-8543, Japan. Received August 16, 1999; accepted November 5, 1999. Cancer Genet Cytogenet 120:80–82 (2000) 2000 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
was not observed. The hemoglobin level was 9.7 g/dL and the white blood cell count was 0.7 ⫻ 109/L with 27% of leukemic promyelocytes. The platelet count was 16 ⫻ 109/L. Coagulation tests revealed a prothrombin time (PT) of 12.7 sec (10.9 ⵑ 12.5 sec), fibrinogen level of 534 mg/dL and fibrin degradation products (FDP) of more than 160 mg/ml. The bone marrow was hyperplastic with 85% atypical promyelocytes, which were packed with numerous azurophilic granules and Auer rods. Leukemic cells were CD13⫹, CD33⫹, CD34⫺, and HLA-DR⫺. A diagnosis of APL was made and all-trans retinoic acid was administered at a daily dose of 60 mg. A complete remission was achieved and chemotherapy including anthracyclines was followed with no recurrence to date. Cytogenetics Chromosome analysis was carried out according to a standard procedure [3]. G-banded chromosomes were designated and described according to ISCN 1995 [4]. Fluorescence In Situ Hybridization Cells kept in fixative at ⫺20⬚C were used for fluorescence in situ hybridization (FISH) analysis. Painting probes of chromosomes 2 and 15 labeled with biotin (Cambio, Cambridge) and of chromosome 17 with digoxigenin (Oncor, Gaithersburg, MD) and a PML/RARA dual-color DNA probe (Vysis, Fujisawa Co. Ltd., Osaka) were used. After hybridization, detection was performed with fluoresceinlabeled avidin/rhodamine-labeled anti-digoxigenin (On-
0165-4608/00/$–see front matter PII S0165-4608(99)00238-1
PML/RARA on 2q in APL with t(2;15;17)
Figure 1 G-banded karyotype of the bone marrow: 45,X,⫺Y,t(2;15;17)(q21;q22;q21). Figure 2 FISH study with painting probes of chromosomes 2 (green), 15 (green), and 17 (red), and with probes of the PML gene (red) and the RARA gene (green). (A) The green signals show chromosome 15 with FITC staining and the red signals show chromosome 17 with rhodamine staining. (B) The green signals show chromosome 2 with FITC staining and the red signals show chromosome 17 with rhodamine staining. (C) Probes of the PML gene (red) appear yellow on chromosome 15 with FITC staining, and PML-RARA fusion signal is seen on a chromosome other than chromosome 15. (D) Chromosome 2 is labeled with FITC (green) and the PML gene (red) is on chromosome 15. PML-RARA fusion signal is seen on the der(2).
81
82
M. Fujishima et al.
cor). The chromosomes were identified by 4⬘-6-diamidino2-phenylindole (DAPI) stain. Fluorescent signals were visualized under a Nikon microscope (Tokyo, Japan) with UV-2A and FITC/rhodamine dual band filters (Nikon). RESULTS Chromosome Analysis The chromosomal aberration was interpreted as 46,XY, t(2;15;17)(q21;q22;q21) [20] (Fig. 1). FISH Analysis The FISH results are illustrated in Figure 2. The chromosome 15 probe (green) hybridized to one chromosome 17 and the chromosome 17 probe (red) hybridized to the other chromosome (Fig. 2A). The chromosome 17 probe (red) hybridized to one chromosome 2 and the chromosome 2 probe (green) hybridized to another chromosome (Fig. 2B). FISH analysis with painting probes supported the karyotypic interpretation of the t(2;15;17). Red signals of the PML gene are on chromosome 15, while the PML/ RARA fusion signal was located on a long chromosome other than chromosome 15 or 17 (Fig. 2C). The PML/ RARA fusion signal was seen on the der(2) (Fig. 2D). These FISH analyses reveal 46,XY,t(2;15;17)(q21;q22;q21) .ish t(2;15;17)(wcp2⫹,PML⫹,RARA⫹,wcp17⫹; wcp15⫹, PML⫺,wcp2⫹; wcp17⫹,RARA⫺,wcp15⫹). DISCUSSION The reciprocal translocation t(15;17) is specifically associated with APL [1] and results in a PML-RARA fusion gene on the der(15). We described an APL patient with t(2;15;17), a complex variant translocation of t(15;17), that was supported by FISH. There have been several complex variant translocations reported in the literature, including t(2;17;15) (q21;q21;q22–24) [2] and t(15;19;17)(q22;p13;q12) [3]. Although the break points of t(2;17;15)(q21;q21;q22–24) [2] were shown to be the same as in our patient, the t(2;17;15) (q21;q21;q22–24) was observed in a second line and was masked by the part of chromosome 2q. We have reported a duplication of BCR-ABL fusion gene on chromosome 9 instead of der(22) in a patient with Ph negative BCR positive CML [5]. Similar events were found in this patient. FISH analysis of our patient showed the fusion PML-RARA gene on the der(2) instead of the der(15). Although the actual order of events could not be confirmed, one hypothetical scheme is shown in Figure 3 namely, to that a standard t(15;17) occurred followed by a translocation between chromosome 2 and the der(15). Since we did not observe a cell with only the t(15;17) in this study, the
Figure 3 Schematic presentation of t(2;15;17) by stepwise translocation. A standard t(15;17) is formed at the first step. As a second step, a translocation between the der(15) and chromosome 2 occurs, PML/RARA fusion gene moves to chromosome 2. possibility that all the events happened at the same time and not in two steps cannot be ruled out. The critical event of APL is the formation of the PML-RARA chimeric gene, regardless of its location in the genome.
REFERENCES 1. Rowley JD, Golomb HM, Dougherty C (1977): 15/17 translocation, a consistent chromosomal change in acute promyelocytic leukaemia. Lancet 1:549–550. 2. Bjerrum OW, Philip P, Pressler T, Tygstrup I (1987): Acute promyelocytic leukemia with t(15;17) and t(2;17;15). Cancer Genet Cytogenet 28:107–111. 3. Saitoh K, Miura I, Kobayashi Y, Kume M, Utsumi S, Takahashi N, Hatano Y, Nimura T, Hashimoto K, Takahashi S, Miura AB (1998): A new variant translocation of t(15;17) in a patient with acute promyelocytic leukemia (M3): t(15;19;17) (q22;p13;q12). Cancer Genet Cytogenet 102:15–18. 4. ISCN (1995): An International System for Human Cytogenetic Nomenclature. F Mitelman, ed. S. Karger, Basel. 5. Takahashi N, Miura I, Ohshima A, Utsumi S, Nimura T, Hashimoto K, Saitoh M, Miura AB (1996): Duplication of chromosome 9 carrying a BCR/ABL chimeric gene in Philadelphia chromosome negative chronic myeloid leukemia. Cancer Genet Cytogenet 89:166–169.