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A New Translocation, t(2;4;12)(p21;q12;p13), in CD7-Positive Acute Myeloid Leukemia
A New Translocation, t(2;4;12)(p21;q12;p13), in CD7-Positive Acute Myeloid Leukemia: A Variant Form of t(4;12) Hiroyuki Hamaguchi, Kaoru Nagata, Katsuya Yamamoto, Masaru Kobayashi, Teruyuki Takashima, and Masafumi Taniwaki
ABSTRACT: We describe a 41-year-old man with CD7-positive acute myeloid leukemia (AML-M0) with trilineage-myelodysplasia. Chromosome analysis of the bone marrow cells showed 46,XY,t(2;4;12) (p21;q12;p13). Cytological and clinical features of our case were quite similar to those of AML with t(4;12)(q11z12;p13). The karyotypic interpretation was confirmed by fluorescence in situ hybridization (FISH) by using the whole-chromosome painting probes specific for chromosomes 2, 4, and 12. FISH analysis with the use of the YAC 936e2 probe, which covers the TEL gene, did not show the split signal, suggesting that a gene other than TEL was involved in the leukemogenesis of the present case. Our case with AML with t(2;4;12)(p21;q12;p13) appears to be the first case of a variant type of AML with t(4;12) (q11z12;p13). © Elsevier Science Inc., 1999. All rights reserved.
INTRODUCTION Acute leukemia with the translocation (4;12)(q11z12;p13) is rare and has common cytological and clinical features [1, 2]. The t(4;12) aberration is seen in primitive or secondary acute myeloid leukemia (AML) in adults or B-lymphoid leukemia in children [2]. Only ten cases of AML with t(4;12) have ever been reported [1–4]. Most showed a trilineage myelodysplasia (TMDS), absent or low myeloperoxidase activity, and a peripheral blood and bone marrow basophilia. The surface markers of the blasts showed positivity for CD7, CD13, CD33, CD34, and HLA-DR, suggesting that the leukemic cells were of immature myeloid stem cell origin. Clinically, AML with t(4;12) has a poor prognosis [1, 2]. In this report, we describe a new case of CD7-positive AML with TMDS carrying a complex translocation, t(2;4;12)(p21; q12; p13), which has common cytological and clinical features with t(4;12) AML. CASE REPORT A 41-year-old man visited another hospital because of fever and shortness of breath on effort on November 25,
From the Department of Hematology, Musashino Red Cross Hospital (H. H., K. N., K. Y.), Tokyo, Japan; SRL, Inc. (M. K.), Tokyo, Japan; and the Third Department of Internal Medicine, Kyoto Prefectural University of Medicine (T. T., M. T.), Kyoto, Japan. Address reprint requests to: Hiroyuki Hamaguchi, M.D., Department of Hematology, Musashino Red Cross Hospital, 1-261 Kyonan-cho, Musashino, Tokyo 180-0023, Japan. Received December 29, 1998; accepted March 30, 1999. Cancer Genet Cytogenet 114:96–99 (1999) Elsevier Science Inc., 1999. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
1996. Because many blastic cells were noted in the peripheral blood, he was referred to our hospital for further evaluation and was admitted on November 27, 1996. Peripheral blood examination showed hemoglobin, 5.9 g/dL, platelets, 7 3 109/L, and white blood cells, 6.9 3 109/L with 61% blasts. Bone marrow examination showed a normocellular marrow with 32.6% blasts, which had folded or clefted nuclei with fine nuclear chromatin. The nuclear and cytoplasmic margins of the blasts were irregular. Some blasts possessed few fine azurophilic granules and vacuoles. The myeloperoxidase reaction of the blasts was faint or negative. The a-naphthylbutyrate esterase and chloroacetate esterase were negative. Marked neutrophilic dysplasias such as a pseudo-Pelger-Huet anomaly and degranulation were seen. Erythroid cells also were dysplastic, having megaloblastoid changes and multinuclear or bizarre nuclear erythroblasts. Megakaryocytes were adequate in number, but they were prominently dysplastic, with micromegakaryocytes and small, round binuclear or separate round multinuclear megakaryocytes. In surface marker analysis, they were positive for CD7 (63.8%), CD13 (70.0%), CD33 (48.6%), CD34 (73.8%), and HLA-DR (89.7%). A diagnosis of CD7-positive AML (M0 in the French–American–British classification) with TMDS was made. The patient was treated by continuous infusion of cytosine arabinoside (100 mg/m2, days 1–7) and idarubicin (12 mg/m2, days 1–3), but remission was not attained. Then, a combination of low doses of cytosine arabinoside (20 mg/m2, days 1–14) and aclarubicin (12 mg/m2, days 1– 4) and recombinant human granulocyte colony-stimulat-
0165-4608/99/$–see front matter PII S0165-4608(99)00060-6
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t(2;4;12)(p21;q12;p13) in AML ing factor (G-CSF; 300 mg, days 1–14) was administered. However, hematological remission was not achieved, and the patient died on February 25, 1997, of aggressive pulmonary aspergillosis after prolonged myelosuppression.
digoxigenin-labeled pBS12 (WCP12) and biotin-labeled yeast artificial chromosome (YAC 936e2) probe containing the TEL gene. FISH analyses with these probes were carried out in 50 metaphases as described previously [6].
MATERIALS AND METHODS
RESULTS
Chromosome Analysis Chromosome analysis was performed on a short-term culture of the cells from the bone marrow by using the Giemsa banding technique. The karyotypes were described according to the International System for Human Cytogenetic Nomenclature [5].
Chromosome Analysis The karyotypes of 20 analyzed metaphases of the bone marrow cells at diagnosis were as follows: 46,XY,t(2;4;12) (p21;q12;p13),del(7) (q22q32)[3]/46,XY,t(2;4;12)(p21;q12; p13), del(7)(q22q32),add(17)(p11)[8]/45,XY,t(2;4;12)(p21; q12;p13), 27,del(7)(q22q32),add(17)(p11)[1]/47,XY,18[6]/ 46,XY[2] (Fig. 1).
Fluorescence In Situ Hybridization Analysis The plasmid DNA libraries pBS2, pBS4, and pBS12, each specific for human chromosomes 2, 4, and 12, were used as a probe for whole-chromosome painting. They were kindly provided by Dr. Joe W. Gray (University of California, San Francisco, CA). pBS4 was labeled with biotin-11-dUTP (Sigma, St. Louis, MO) and detected with fluorescein isothiocyanate (FITC). pBS12 was labeled with digoxigenin-11-dUTP (Boehringer, Germany) and detected with tetramethylrhodamine (TRITC). Equal amounts of biotin-11dUTP- and digoxigenin-11-dUTP-labeled pBS2 were mixed and detected with FITC and TRITC. The same metaphases were also counterstained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma). The involvement of the TEL gene, located on 12p13, was studied by two-color fluorescence in situ hybridization (FISH) analysis by using
FISH Analysis To confirm the chromosomal translocation, we performed a three-color FISH analysis with chromosome-specific DNA libraries for chromosomes 2, 4, and 12 on the metaphase spreads (Fig. 2). The red signals show digoxigenin staining of chromosome 12, the green signals show biotin staining of chromosome 4, and the yellow signals show biotin and digoxigenin staining of chromosome 2. The probes clearly painted two derivative chromosomes, der(4)t(2;4)(p21;q12) and der(12)t(4;12)(q12;p13), as well as normal chromosomes 4 and 12 (Fig. 2A and B). Two yellow signals show normal chromosome 2 and der(2) t(2;12)(p21;p13), which has a short p arm as a result of the translocation. However, the red signal on the derivative chromosome 2 is hardly seen, because it is masked by the
Figure 1 G-banded karyotype of the patient: 46,XY,t(2;4;12)(p21;q12;p13),del(7)(q22q32),add(17)(p11). Derivative chromosomes are indicated by arrows. Other abnormal chromosomes are indicated by arrowheads.
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Figure 2 (A) Three-color FISH analysis with painting probes for chromosomes 2 (pBS2; yellow), 4 (pBS4; green), and 12 (pBS12; red). Arrows indicate (1) der(2)t(2;12)(p21;p13), (2) der(4)t(2;4)(p21;q12), and (3) der(12)t(4;12) (q12;p13). (B) The DAPI staining picture of the corresponding metaphase cell. (C) Two-color FISH analysis with pBS12 (WCP12; red) and YAC936e2 clone (green) containing the TEL gene on metaphase spread. YAC936e2 on chromosome 12 was detected as a yellow signal from green on red. Arrows indicate (1) YAC 936e2 signal on normal chromosome 12 and (2) YAC 936e2 signal on der(12)t(4;12). (D) DAPI staining of the same metaphase as (c).
yellow signal. We next investigated the involvement of the TEL gene by two-color FISH with the use of the biotinlabeled YAC 936e2 probe (green), which includes the TEL gene and the digoxigenin-labeled pBS12 probe (red). Twocolor FISH analysis showed that the 936e2 signals (detected as a yellow signal as a result of green on red signal) were on the normal chromosome 12 and on the derivative chromosome 12 in equal intensity (Fig. 2B). DISCUSSION On the basis of the results of cytogenetic and FISH analyses, we interpreted at the karyotype to be 46,XY,t(2;4;12) (2pter→2p21::4q12→4pter;4qter→4q12::12p13→12qter; 2qter→2p21::12p13→12pter). This is the first report of such a three-way translocation involving chromosomes 2, 4, and 12 in AML. The translocation (4;12) is a rare but nonrandom aberration, and ten adult cases of t(4;12) AML have been reported [1–4]. The patients with AML with t(4;12) have common biological and clinical features. They have a three-lineage dysplasia, absent or low myeloperoxi-
dase activity, retention of platelets in the peripheral blood and megakaryocytes in the bone marrow, and a peripheral blood and bone marrow basophilia [1–3]. The immunophenotype showed positivity for CD7, CD13, CD33, CD34, and HLA-DR, which suggested that this translocation may have occurred in an early myeloid stem cell. CD7 was positive in four of five cases examined. Clinically, t(4;12) AML is difficult to treat by the usual chemotherapy, and prognosis is unfavorable. The present case had morphological and clinical features quite similar to those of AML with t(4;12)(q11,12;p13) described by Harada et al. [1, 2]. Our case also had a three-lineage dysplasia, low myeloperoxidase activity, retention of megakaryocytes in the bone marrow, and positivity for CD7, CD13, CD33, CD34, and HLA-DR. Furthermore, the patient was refractory to initial remission-induction chemotherapy and died of aggressive pulmonary aspergillosis after prolonged myelosuppression. Thus, the t(2;4;12) appeared to be a variant form of t(4;12). Our case also suggests that the der(12) t(4;12) is essential for leukemogenesis of t(4;12) AML. The translocation breakpoints at 12p13 were found to
t(2;4;12)(p21;q12;p13) in AML be clustered within the sequences in the 1.39-Mb YAC that contained the TEL gene affected in various malignant hematologic diseases [7, 8]. The TEL gene, one of the ETS family and located on band 12p13, has been shown to be fused to the PDGFR-b gene in chronic myelomonocytic leukemia with t(5;12)(q33;p13) [9], fused with the MN1 gene in myeloproliferative disorders with t(12;22)(p13; q11) [10] and disrupted in myelodysplastic syndrome with t(3;12)(q26;p13) [11]. The involvement of the TEL gene was studied in three cases of AML with t(4;12) by using FISH and pulsed-field gel electrophoresis [2, 4]. The results showed that the breakpoints of 12p13 in t(4;12) AML were located within or near the TEL gene [2, 4]. We also investigated the involvement of the TEL gene by using two-color FISH with YAC936e2, which includes the TEL gene. In the present case, however, a split signal was not detected, and YAC936e2 hybridized only to the normal and rearranged chromosome 12, indicating that the translocation breakpoint was telomeric to YAC936e2. Considering these results, we suggest that the 12p13 breakpoints in the t(4;12) AML may be heterogenous and that the TEL gene is not always involved in t(4;12) AML. Heterogeneity in the breakpoints involving band 12p13 in various hematologic malignancies was reported [8, 12]. Therefore, some unidentified genes other than TEL may be involved in the leukemogenesis of t(4;12) AML. A number of genes have been mapped to the band 4q12. These genes include an insulin growth-factor-like binding protein gene (mac25) [13], platelet-derived growth factor alpha gene (PDGFRA) [14], alpha-fetoprotein gene (AFP) [15], and a beta-sarcoglycan gene that is involved in limb-girdle muscular dystrophy [16]. However, whether these genes are involved in leukemogenesis of t(4;12) AML is unknown. Further molecular study would be required to elucidate these problems. REFERENCES 1. Harada H, Asou H, Kyo T, Asaoku H, Iwato K, Dohy H, Oda K, Harada Y, Kita K, Kamada N (1995): A specific chromosome abnormality of t(4;12)(q11–12;p13) in CD71 acute leukemia. Br J Haematol 90:850–854. 2. Harada H, Harada Y, Eguchi M, Dohy H, Kamada N (1997): Characterization of acute leukemia with t(4;12). Leuk Lymphoma 25:47–53. 3. Ma SK, Lie AKW, Au WY, Wan TSK, Chan LC (1997): CD71 acute myeloid leukaemia with “mature lymphoid” blast morphology, marrow basophilia and t(4;12)(q12;p13). Br J Haematol 97:978–980. 4. Sainty D, Arnoulet C, Mozziconacci MJ, De Pina JJ, Garnotel E, Lafage-Pochitaloff M (1997): t(4;12)(q11;p13) in a CD7-negative acute leukaemia. Br J Haematol 96:210–212.
99 5. ISCN (1995): An International System for Human Cytogenetic Nomenclature. F Mitelman, ed. S. Karger, Basel. 6. Yamamoto K, Hamaguchi H, Nagata K, Kobayashi M, Tanimoto F, Taniwaki M (1997): Establishment of a novel human acute myeloblastic leukemia cell line (YNH-1) with t(16;21), t(1;16) and 12q13 translocations. Leukemia 11:599–608. 7. Kobayashi H, Montgomery KM, Bohlander SK, Adra CN, Lim BL, Kucherlapati RS, Donis-Keller H, Holt MS, Le Beau MM, Rowley JD (1994): Fluorescence in situ hybridization mapping of translocations and deletions involving the short arm of human chromosome 12 in malignant hematologic disease. Blood 84:3473–3482. 8. Wlodarska I, Starza RL, Baens M, Dierlamm J, Uyttebroeck A, Selleslag D, Francine A, Mecucci C, Hagemeijer A, Van den Berghe H, Marynen P (1998): Fluorescence in situ hybridization characterization of new translocations involving TEL (ETV6) in a wide spectrum of hematologic malignancies. Blood 91:1399–1406. 9. Golub TR, Barker GF, Lovett M, Gilliland DG (1994): Fusion of PDGF recepter b to a novel ets-like gene, TEL, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation. Cell 77:307–316. 10. Buijs A, Sherr SS, van Baal S, van Bezouw S, van der Plas D, van Kessel AG, Riegman P, Deprez RL, Zwarthoff E, Hagemeijer A, Grosveld G (1995): Translocation (12;22)(p13;q11) in myeloproliferative disorders results in fusion of the ETS-like TEL gene on 12p13 to the MN1 gene on 22q11. Oncogene 10:1511–1519. 11. Raynaud SD, Baens M, Grosgeorge J, Rodgers K, Reid CDL, Dainton M, Dyer M, Fuzibet JG, Gratecos N, Taillan B, Ayraud N, Marynen P (1996): Fluorescence in situ hybridization analysis of t(3;12)(q26;p13): a recurring chromosomal abnormality involving the TEL gene (ETV6) in myelodysplastic syndromes. Blood 88:682–689. 12. Sato Y, Bohlander SK, Kobayashi H, Reshmi S, Suto Y, Davis EM, Espinosa R, III, Hoopes R, Montgomery KT, Kucherlapati RS, Le Beau MM, Rowley JD (1997): Heterogeneity in the breakpoints in balanced rearrangements involving band 12p13 in hematologic malignancies identified by fluorescence in situ hybridization: TEL (ETV6) is involved in only one half. Blood 90:4886–4893. 13. Swisshelm K, Ryan K, Tsuchiya K, Sager R (1995): Enhanced expression of an insulin growth factor-like binding protein (mac 25) in senescent human mammary epithelial cells and induced expression with retinoic acid. Proc Natl Acad Sci USA 92:4472–4476. 14. Spritz RA, Strunk KM, Lee ST, Lu-Kuo JM, Ward DC, Le Paslier D, Altherr MR, Dorman TE, Moir DT (1994): A YAC contig spanning a cluster of human type III receptor protein tyrosine kinase genes (PDGFRA-KIT-KDR) in chromosome segment 4q12. Genomics 22:431–436. 15. Leon M, Kew MC (1996): Loss of heterozygosity in chromosome 4q12–q13 in hepatocellular carcinoma in southern African blacks. Anticancer Res 16:349–351. 16. Lim LE, Duclos F, Broux O, Bourg N, Sunada Y, Allamand V, Meyer J, Richard I, Moomaw C, Slaughter C (1995): Betasarcoglycan: characterization and role in limb-girdle muscular dystrophy linked to 4q12. Nat Genet 11:257–265.
ABSTRACT: We describe a 41-year-old man with CD7-positive acute myeloid leukemia (AML-M0) with trilineage-myelodysplasia. Chromosome analysis of the bone marrow cells showed 46,XY,t(2;4;12) (p21;q12;p13). Cytological and clinical features of our case were quite similar to those of AML with t(4;12)(q11z12;p13). The karyotypic interpretation was confirmed by fluorescence in situ hybridization (FISH) by using the whole-chromosome painting probes specific for chromosomes 2, 4, and 12. FISH analysis with the use of the YAC 936e2 probe, which covers the TEL gene, did not show the split signal, suggesting that a gene other than TEL was involved in the leukemogenesis of the present case. Our case with AML with t(2;4;12)(p21;q12;p13) appears to be the first case of a variant type of AML with t(4;12) (q11z12;p13). © Elsevier Science Inc., 1999. All rights reserved.
INTRODUCTION Acute leukemia with the translocation (4;12)(q11z12;p13) is rare and has common cytological and clinical features [1, 2]. The t(4;12) aberration is seen in primitive or secondary acute myeloid leukemia (AML) in adults or B-lymphoid leukemia in children [2]. Only ten cases of AML with t(4;12) have ever been reported [1–4]. Most showed a trilineage myelodysplasia (TMDS), absent or low myeloperoxidase activity, and a peripheral blood and bone marrow basophilia. The surface markers of the blasts showed positivity for CD7, CD13, CD33, CD34, and HLA-DR, suggesting that the leukemic cells were of immature myeloid stem cell origin. Clinically, AML with t(4;12) has a poor prognosis [1, 2]. In this report, we describe a new case of CD7-positive AML with TMDS carrying a complex translocation, t(2;4;12)(p21; q12; p13), which has common cytological and clinical features with t(4;12) AML. CASE REPORT A 41-year-old man visited another hospital because of fever and shortness of breath on effort on November 25,
From the Department of Hematology, Musashino Red Cross Hospital (H. H., K. N., K. Y.), Tokyo, Japan; SRL, Inc. (M. K.), Tokyo, Japan; and the Third Department of Internal Medicine, Kyoto Prefectural University of Medicine (T. T., M. T.), Kyoto, Japan. Address reprint requests to: Hiroyuki Hamaguchi, M.D., Department of Hematology, Musashino Red Cross Hospital, 1-261 Kyonan-cho, Musashino, Tokyo 180-0023, Japan. Received December 29, 1998; accepted March 30, 1999. Cancer Genet Cytogenet 114:96–99 (1999) Elsevier Science Inc., 1999. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
1996. Because many blastic cells were noted in the peripheral blood, he was referred to our hospital for further evaluation and was admitted on November 27, 1996. Peripheral blood examination showed hemoglobin, 5.9 g/dL, platelets, 7 3 109/L, and white blood cells, 6.9 3 109/L with 61% blasts. Bone marrow examination showed a normocellular marrow with 32.6% blasts, which had folded or clefted nuclei with fine nuclear chromatin. The nuclear and cytoplasmic margins of the blasts were irregular. Some blasts possessed few fine azurophilic granules and vacuoles. The myeloperoxidase reaction of the blasts was faint or negative. The a-naphthylbutyrate esterase and chloroacetate esterase were negative. Marked neutrophilic dysplasias such as a pseudo-Pelger-Huet anomaly and degranulation were seen. Erythroid cells also were dysplastic, having megaloblastoid changes and multinuclear or bizarre nuclear erythroblasts. Megakaryocytes were adequate in number, but they were prominently dysplastic, with micromegakaryocytes and small, round binuclear or separate round multinuclear megakaryocytes. In surface marker analysis, they were positive for CD7 (63.8%), CD13 (70.0%), CD33 (48.6%), CD34 (73.8%), and HLA-DR (89.7%). A diagnosis of CD7-positive AML (M0 in the French–American–British classification) with TMDS was made. The patient was treated by continuous infusion of cytosine arabinoside (100 mg/m2, days 1–7) and idarubicin (12 mg/m2, days 1–3), but remission was not attained. Then, a combination of low doses of cytosine arabinoside (20 mg/m2, days 1–14) and aclarubicin (12 mg/m2, days 1– 4) and recombinant human granulocyte colony-stimulat-
0165-4608/99/$–see front matter PII S0165-4608(99)00060-6
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t(2;4;12)(p21;q12;p13) in AML ing factor (G-CSF; 300 mg, days 1–14) was administered. However, hematological remission was not achieved, and the patient died on February 25, 1997, of aggressive pulmonary aspergillosis after prolonged myelosuppression.
digoxigenin-labeled pBS12 (WCP12) and biotin-labeled yeast artificial chromosome (YAC 936e2) probe containing the TEL gene. FISH analyses with these probes were carried out in 50 metaphases as described previously [6].
MATERIALS AND METHODS
RESULTS
Chromosome Analysis Chromosome analysis was performed on a short-term culture of the cells from the bone marrow by using the Giemsa banding technique. The karyotypes were described according to the International System for Human Cytogenetic Nomenclature [5].
Chromosome Analysis The karyotypes of 20 analyzed metaphases of the bone marrow cells at diagnosis were as follows: 46,XY,t(2;4;12) (p21;q12;p13),del(7) (q22q32)[3]/46,XY,t(2;4;12)(p21;q12; p13), del(7)(q22q32),add(17)(p11)[8]/45,XY,t(2;4;12)(p21; q12;p13), 27,del(7)(q22q32),add(17)(p11)[1]/47,XY,18[6]/ 46,XY[2] (Fig. 1).
Fluorescence In Situ Hybridization Analysis The plasmid DNA libraries pBS2, pBS4, and pBS12, each specific for human chromosomes 2, 4, and 12, were used as a probe for whole-chromosome painting. They were kindly provided by Dr. Joe W. Gray (University of California, San Francisco, CA). pBS4 was labeled with biotin-11-dUTP (Sigma, St. Louis, MO) and detected with fluorescein isothiocyanate (FITC). pBS12 was labeled with digoxigenin-11-dUTP (Boehringer, Germany) and detected with tetramethylrhodamine (TRITC). Equal amounts of biotin-11dUTP- and digoxigenin-11-dUTP-labeled pBS2 were mixed and detected with FITC and TRITC. The same metaphases were also counterstained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma). The involvement of the TEL gene, located on 12p13, was studied by two-color fluorescence in situ hybridization (FISH) analysis by using
FISH Analysis To confirm the chromosomal translocation, we performed a three-color FISH analysis with chromosome-specific DNA libraries for chromosomes 2, 4, and 12 on the metaphase spreads (Fig. 2). The red signals show digoxigenin staining of chromosome 12, the green signals show biotin staining of chromosome 4, and the yellow signals show biotin and digoxigenin staining of chromosome 2. The probes clearly painted two derivative chromosomes, der(4)t(2;4)(p21;q12) and der(12)t(4;12)(q12;p13), as well as normal chromosomes 4 and 12 (Fig. 2A and B). Two yellow signals show normal chromosome 2 and der(2) t(2;12)(p21;p13), which has a short p arm as a result of the translocation. However, the red signal on the derivative chromosome 2 is hardly seen, because it is masked by the
Figure 1 G-banded karyotype of the patient: 46,XY,t(2;4;12)(p21;q12;p13),del(7)(q22q32),add(17)(p11). Derivative chromosomes are indicated by arrows. Other abnormal chromosomes are indicated by arrowheads.
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Figure 2 (A) Three-color FISH analysis with painting probes for chromosomes 2 (pBS2; yellow), 4 (pBS4; green), and 12 (pBS12; red). Arrows indicate (1) der(2)t(2;12)(p21;p13), (2) der(4)t(2;4)(p21;q12), and (3) der(12)t(4;12) (q12;p13). (B) The DAPI staining picture of the corresponding metaphase cell. (C) Two-color FISH analysis with pBS12 (WCP12; red) and YAC936e2 clone (green) containing the TEL gene on metaphase spread. YAC936e2 on chromosome 12 was detected as a yellow signal from green on red. Arrows indicate (1) YAC 936e2 signal on normal chromosome 12 and (2) YAC 936e2 signal on der(12)t(4;12). (D) DAPI staining of the same metaphase as (c).
yellow signal. We next investigated the involvement of the TEL gene by two-color FISH with the use of the biotinlabeled YAC 936e2 probe (green), which includes the TEL gene and the digoxigenin-labeled pBS12 probe (red). Twocolor FISH analysis showed that the 936e2 signals (detected as a yellow signal as a result of green on red signal) were on the normal chromosome 12 and on the derivative chromosome 12 in equal intensity (Fig. 2B). DISCUSSION On the basis of the results of cytogenetic and FISH analyses, we interpreted at the karyotype to be 46,XY,t(2;4;12) (2pter→2p21::4q12→4pter;4qter→4q12::12p13→12qter; 2qter→2p21::12p13→12pter). This is the first report of such a three-way translocation involving chromosomes 2, 4, and 12 in AML. The translocation (4;12) is a rare but nonrandom aberration, and ten adult cases of t(4;12) AML have been reported [1–4]. The patients with AML with t(4;12) have common biological and clinical features. They have a three-lineage dysplasia, absent or low myeloperoxi-
dase activity, retention of platelets in the peripheral blood and megakaryocytes in the bone marrow, and a peripheral blood and bone marrow basophilia [1–3]. The immunophenotype showed positivity for CD7, CD13, CD33, CD34, and HLA-DR, which suggested that this translocation may have occurred in an early myeloid stem cell. CD7 was positive in four of five cases examined. Clinically, t(4;12) AML is difficult to treat by the usual chemotherapy, and prognosis is unfavorable. The present case had morphological and clinical features quite similar to those of AML with t(4;12)(q11,12;p13) described by Harada et al. [1, 2]. Our case also had a three-lineage dysplasia, low myeloperoxidase activity, retention of megakaryocytes in the bone marrow, and positivity for CD7, CD13, CD33, CD34, and HLA-DR. Furthermore, the patient was refractory to initial remission-induction chemotherapy and died of aggressive pulmonary aspergillosis after prolonged myelosuppression. Thus, the t(2;4;12) appeared to be a variant form of t(4;12). Our case also suggests that the der(12) t(4;12) is essential for leukemogenesis of t(4;12) AML. The translocation breakpoints at 12p13 were found to
t(2;4;12)(p21;q12;p13) in AML be clustered within the sequences in the 1.39-Mb YAC that contained the TEL gene affected in various malignant hematologic diseases [7, 8]. The TEL gene, one of the ETS family and located on band 12p13, has been shown to be fused to the PDGFR-b gene in chronic myelomonocytic leukemia with t(5;12)(q33;p13) [9], fused with the MN1 gene in myeloproliferative disorders with t(12;22)(p13; q11) [10] and disrupted in myelodysplastic syndrome with t(3;12)(q26;p13) [11]. The involvement of the TEL gene was studied in three cases of AML with t(4;12) by using FISH and pulsed-field gel electrophoresis [2, 4]. The results showed that the breakpoints of 12p13 in t(4;12) AML were located within or near the TEL gene [2, 4]. We also investigated the involvement of the TEL gene by using two-color FISH with YAC936e2, which includes the TEL gene. In the present case, however, a split signal was not detected, and YAC936e2 hybridized only to the normal and rearranged chromosome 12, indicating that the translocation breakpoint was telomeric to YAC936e2. Considering these results, we suggest that the 12p13 breakpoints in the t(4;12) AML may be heterogenous and that the TEL gene is not always involved in t(4;12) AML. Heterogeneity in the breakpoints involving band 12p13 in various hematologic malignancies was reported [8, 12]. Therefore, some unidentified genes other than TEL may be involved in the leukemogenesis of t(4;12) AML. A number of genes have been mapped to the band 4q12. These genes include an insulin growth-factor-like binding protein gene (mac25) [13], platelet-derived growth factor alpha gene (PDGFRA) [14], alpha-fetoprotein gene (AFP) [15], and a beta-sarcoglycan gene that is involved in limb-girdle muscular dystrophy [16]. However, whether these genes are involved in leukemogenesis of t(4;12) AML is unknown. Further molecular study would be required to elucidate these problems. REFERENCES 1. Harada H, Asou H, Kyo T, Asaoku H, Iwato K, Dohy H, Oda K, Harada Y, Kita K, Kamada N (1995): A specific chromosome abnormality of t(4;12)(q11–12;p13) in CD71 acute leukemia. Br J Haematol 90:850–854. 2. Harada H, Harada Y, Eguchi M, Dohy H, Kamada N (1997): Characterization of acute leukemia with t(4;12). Leuk Lymphoma 25:47–53. 3. Ma SK, Lie AKW, Au WY, Wan TSK, Chan LC (1997): CD71 acute myeloid leukaemia with “mature lymphoid” blast morphology, marrow basophilia and t(4;12)(q12;p13). Br J Haematol 97:978–980. 4. Sainty D, Arnoulet C, Mozziconacci MJ, De Pina JJ, Garnotel E, Lafage-Pochitaloff M (1997): t(4;12)(q11;p13) in a CD7-negative acute leukaemia. Br J Haematol 96:210–212.
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