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Trisomy 11 and a Complex t(11;11;22) in a Patient with Acute Myelomonocytic Leukemia (AML-M4) Following Myelodysplasia (MDS)
Trisomy 11 and a Complex t(11;11;22) in a Patient with Acute Myelomonocytic Leukemia (AML-M4) Following Myelodysplasia (MDS): A Cytogenetic Study of a Mechanism of Leukemogenesis P. Bernasconi, P. M. Cavigliano, M. Boni, L. Malcovati, C. Astori, C. Castagnola, G. Pagnucco, L. Vanelli, S. Calatroni, M. Caresana, M. Lazzarino, and C. Bernasconi
ABSTRACT: We describe a 73-year-old man diagnosed with acute myelomonocytic leukemia (AMLM4) following myelodysplasia with trisomy 11 and with a t(11;11;22) . This is the first case with both abnormalities present in the same cells and with the t(11;11;22) involving a chromosome 11 already duplicated at 11q23. This band contains the MLL gene that undergoes partial tandem duplication in patients with 111, which is “promiscuous,” being translocated with a large number of genetic partners. Our patient had a complex karyotype that was completely defined by in situ hybridization. This technique demonstrated that the t(11;11;22) derivative with a duplication of band 11q23 carried from three to four copies of MLL. Two copies of the gene were close to each other and centromeric to the breakpoint region. Therefore, a partial tandem duplication of the MLL gene might have happened before the occurrence of t(11;11;22). Considering the associated chromosome defects, the monosomy for the long arm of chromosome 7, due to an unbalanced translocation t(7;17), further underlines the possibility that a partial tandem duplication of the MLL gene might have taken place. © Elsevier Science Inc., 1999. All rights reserved. INTRODUCTION Recurrent chromosome abnormalities involving band 11q23 have been described in various hematologic disorders. This chromosome band can be joined to one of 25 other chromosomal regions and, recently, 10 further new partner chromosome sites were identified [1]. The majority of these translocations cause the rearrangement of the MLL gene, mapped at band 11q23 [2, 3]. Breakpoint clusters fall within exons 5 to 10 of the MLL gene [4]. All these translocations lead to the formation of a chimeric gene that is part MLL and part of a gene on the reciprocal chromosome. Recently, a different MLL rearrangement, consisting of partial tandem duplication of exons 2 through 6,
From the Istituto di Ematologia, Università di Pavia, Divisione di Ematologia, Policlinico San Matteo IRCCS, 27100 Pavia, Italy. Address reprint requests to: Dr. Paolo Bernasconi, Divisione di Ematologia, Policlinico San Matteo IRCCS, Piazzale Golgi N.5, 27100 Pavia, Italy. Received February 3, 1999; accepted May 25, 1999. Cancer Genet Cytogenet 116:111–118 (2000) Elsevier Science Inc., 1999. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
localized in the critical region of the fused MLL partner transcript, was reported in AML/MDS (acute myelomonocytic leukemia/myelodysplasia) patients with an isolated trisomy 11 (111) or with a normal karyotype [5]. Therefore, the fusion of a part of a putative proto-oncogene with itself was proposed as a novel mechanism for leukemogenesis. Clinically, 11q23 translocations or deletions are associated with myelomonocytic, monoblastic, or biphenotypic leukemia, showing a high blast cell count on presentation and a poor prognosis [6, 7], whereas 111 is associated with a stem/progenitor cell leukemia, having a poor response to standard chemotherapeutic regimens and a dismal prognosis [8]. We report a case of AML-M4 evolved from MDS, without previous exposure to topoisomerase II inhibitors, carrying a complex karyotype. This karyotype showed an unbalanced t(Y;5), leading to the almost complete loss of a chromosome 5 long arm and of a part of the Y chromosome between Ypter→Yq11, an unbalanced t(7;17), resulting in loss of a chromosome 7 long arm, trisomy 11, and a translocation t(11;11;22), leading to three derivatives.
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MATERIALS AND METHODS
CYTOGENETICS AND FISH RESULTS
Cytogenetic and Fluorescence In Situ Hybridization Cytogenetic analysis was carried out on bone marrow cells at diagnosis by using a trypsin-Giemsa banding technique. Metaphase cells were examined from short-term unstimulated cultures. Chromosomal abnormalities were defined according to ISCN [9]. Fluorescence in situ hybridization (FISH) was performed on cytogenetic preparations. At first we used three probes, all specific for chromosome 11: one centromeric probe, one painting probe, and one MLL (11q23) DNA probe. The last probe is specific for genomic sequences including the MLL locus. It detects the majority of rearrangements at the MLL gene locus by showing a large spot on the normal chromosome 11 and two smaller signals, one on the abnormal chromosome 11 and one at the site of the translocation. The 11q23 DNA probe does not detect translocations involving a 39 deletion of the MLL gene region. The centromeric probe was biotinylated (green spot); both of the other two were digoxigenated (red signals). Then, we employed digoxigenin-labelled painting probes for chromosomes 5, 17, and 22 (red signals). Two alphoid biotinylated probes for numbers 5 and 22 also were used (green spots). All these probes were commercially obtained (Oncor Inc., Gaithersburg, MD). Hybridization was carried out according to the manufacturer’s guidelines.
The karyotype was initially defined as: 47,X,2Y,del(5) (q13q33),der(7)(7pter→7q11<17q11→17qter), dup(11) (pter→ q23
CASE REPORT E. G., a 73-year-old man, never exposed to topoisomerase II inhibitors or to other genotoxic agents, came to us for observation because of fatigue and fever in November 1996. On physical examination, hepatomegaly (2 cm) was found. The patient’s blood count was as follows: hemoglobin, 8.1g/dl; white blood cell (WBC) count, 18.9 3 109/L (differential: neutrophils, 12%; lymphocytes, 8%; myelocytes, 12%; myeloblasts, 68%); and platelet count, 35.0 3 109/L. A bone marrow aspirate showed the almost exclusive presence (70%) of blasts, 22% with a monocytic appearance. Moreover, the morphologic features were suggestive of an underlying myelodysplasia because internuclear bridging in erythroid precursors, neutrophil hypogranulation, and micromegakaryocytes were noticed. Myeloperoxidase was positive in 88% of the blast cells, acetate esterase in 80%, and a-naphtyl acetate esterase, inhibited with NaF, in 42% of blast cells. The immunophenotyping of peripheral blood blast cells showed: HLA-DR (34%), CD34 (31%), CD33 (98%), CD13 (98%), CD14 (54%), CD15 (53%), CD11b (94%), and CD11c (89%). Serum lysozyme level was 82.3 mL/mL (normal value less than 10 mL/mL). The patient was classified as AML-M4, evolved from MDS. The chemotherapy regimen consisted of only 6-thioguanine, 40 mg in two daily doses for 5 days every 14 days, because of age and poor clinical conditions. After three cycles, there was a progressive rise in the WBC count and Ara-C, 80 mg/m2, in two daily doses for 5 days was started. The patient did not respond to this second treatment and died of sepsis.
Translocation (11;11;22) in a Case of MDS/AML
Figure 1 FISH definition of the t(11;11;22). (a) Hybridization with a chromosome 11 alphoid probe (green spots) on a cell with der(11)(;11p13→11q13<22q11→22qter)(mar3) (→) and with der(11)(11pter→11q23<11q23→ 11q25< 11q13→11qter) (----x): three copies of chromosome 11 are identified. (b) Hybridization with the MLL DNA probe (red signals) on the same metaphase, counterstained with DAPI: one spot is on the normal chromosome 11, three spots on der(11)(11pter→11q23<11q23→11q25< 11q13→11qter) (----x), none on the other derivative (mar3) (→). (c) Hybridization with chromosome 11 painting probe (red signals) on the same cell: the normal chromosome 11 and der(11)(11pter→ 11q23<11q23→11q25<11q13→11qter) (----x) are completely stained; der(11)(;11p13→ 11q13<22q11→ 22qter)(mar3) (→), mar1, and mar2 are only partly covered by the probe. (a9 through c9) The same hybridization steps on a cell with r(11) (.). (a0 through c0) The same hybridization steps on a cell with dic(11;11;22) (⇒). (c0) The dicentric is only partly stained by the painting probe. (d) Hybridization with a chromosome 22 painting probe (red signals) in a cell carrying der(11)(;11p13→11q13< 22q11→22qter) (→) and the other der(11) (----x) with only one centromere: one spot is on normal 22; the other one is on der(11)(;11p13→ 11q13<22q11→22qter) (→). (d9) Hybridization with the same probe on a cell with dic(11;11;22): one signal is on normal 22; the other one is on dic(11;11;22) (⇒). (e) FISH with chromosome 22 painting and alphoid probe (red and green spots) in a cell with dic(11;11;22) (⇒): one red signal is on the normal 22, and the other one is on dic(11;11;22) (⇒); one green spot is on the normal 22, and the other one identifies der(22)(mar4), consisting of only the centromere (c).
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Figure 2 FISH definition of the t(Y;5) and the t(7;17). (a) A DAPI-stained metaphase: the chromosome with the Y heterochromatic region is indicated (→). (a9) Hybridization with a 5a probe (green spots) on the preceding metaphase: one of the signals is on a chromosome with Y heterochromatic region (→), the other spot is on normal 5, and the last two are on chromosomes 1, inasmuch as the probe cross hybridizes with the centromere of this chromosome. (b) A DAPI-stained metaphase: the chromosome with Y heterochromatic region is indicated (→). (b9) Hybridization with a chromosome 5 and 22 painting probe (red signals) on the preceding metaphase: one signal is on a chromosome with Y heterochromatic region (→), and the largest one is on normal chromosome 5. The other spots identify normal 22 and dic(11;11;22) (⇒). (c) FISH with a chromosome 17 painting probe (red signals): three signals are observed, one corresponding to normal 17, one to der(7) (→), and one to mar5 (----x).
der(22)(22pter→22q11;),1der(?)t(?;11)(wcp111,cen112, MLL2),1der(?)t(?;11)(wcp111,cen112,MLL2)[5] (Fig. 4). DISCUSSION This is the first patient presenting with 111 and with a reciprocal t(11;11;22) in the same cells. Trisomy 11 has been reported in MDS, in AML, and in Philadelphia chromosome-negative chronic granulocytic leukemia [11, 12]. This numerical chromosome change is detected in about 1% of MDS/AML and does not correlate with any specific French–American–British subgroup of MDS/AML; probably, it marks a pluripotent stem cell disorder being discovered in primary as well as in secondary MDS/AML [8]. It has been suggested [11] that 111 is especially associated with myelomonocytic proliferation. The immunophenotyping of our patient is in line with that suggestion, because his blast cells expressed antigens of early myeloid progenitor cells (CD34, HLA-DR), of committed myeloid cells (CD15, CD13, CD33), and of monocytic cells (CD11b, CD11c). The expression of the first five antigens may be re-
lated to 111, whereas that of the last two may be related to the t(11;11). In fact, 11q23 translocations are typically associated with a M4-M5 cytotype. Moreover, the two t(11;11) cases reported in the literature [13, 14] also have been classified as M4 and M5b, respectively. The age range for 1 11 is from ,1 to 82 years, and a review of the literature indicates that from 30% to 50% of 111 patients have an underlying MDS, which frequently evolves into AML [8, 15]. A preleukemia phase was also present in a 13-year-old female with a t(11;11) and an MLL rearrangement [14]. Our patient, a 73-year-old man, probably had an unrecognized myelodysplasia before AML development because, at our observation, a trilineage dysplasia was still present in his marrow aspirate. Conventional cytogenetics allowed us to detect only the largest product of the t(11;11;22), which was interpreted as a dup(11). FISH, on the contrary, succeeded in completely defining the karyotype. At first, a chromosome 11 centromeric probe demonstrated that almost every cell (97%) carried 111. The successive use of the MLL probe showed this gene localized in a single copy on the normal chromo-
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Figure 3 Schematic representation of normal chromosomes 5 and Y and of der(5)(5pter→5q11
some 11 and in three copies on dup(11) with one centromere. Because the two red signals on the dup(11) resided close to each other and centromeric to the breakpoint regions, we suggest that this rearranged chro-
mosome 11 had had a duplication of band 11q23 before the occurrence of the t(11;11;22) (Fig. 5). This same chromosome carried a telomeric MLL signal, derived from the other product of the translocation der(11)(;11p13→ 11q13<22q11→22qter), which, in fact, did not show any spot due to MLL. Therefore, we suggest that the translocation took place between one chromosome 11 (already duplicated at band 11q23 and probably deleted within band 11q25), the other chromosome 11 (already deleted at 11p13, with a breakpoint at 11q13), and one chromosome 22 (deleted at band q11). The first der(11) received no material from the other two, the second der(11) obtained band 22q11→qter from chromosome 22, and the der(22) consisted of only the centromere. We do not know whether band 11q25 of der(11)(11pter→11q23<11q23→ 11q25<11q13→11qter) was lost; however, no material from chromosome 11 was present on the der(22) (Fig. 5). We did not study the MLL gene from a molecular point of view, and therefore we cannot establish whether this gene underwent a partial tandem duplication. Recently, it was reported that one attractive mechanism for this molecular defect involves a t(11;11)(q23;q23), occurring within the MLL gene locus [5]. In the present case, we cannot rule out the possibility that a breakpoint within band 11q23 might have happened, just when this same band underwent duplication (hypothesized step, Fig. 5). However, in our patient, genes residing within chromosome 11 short arm, too, might have been important for the development of his disease. In fact, the t(11;11;22) involved a chromosome 11 already deleted at p13. In five cells, we observed the absence of der(11)(;11p13→ 11q13<22q11→22qter) and the presence of dic(11;11;22).
Figure 4. Karyotype of a trypsin-Giemsa banded metaphase. Rearranged chromosomes are indicated by arrows.
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Figure 5 Schematic representation of the t(11;11;22). der(11)(;11p13→11q13<22q11→22qter)der(11)(11pter→ 11q23<11q23→11q25<11q13→11qter) and der(22)(22pter→22q11;) are shown. The hypothesized steps of the rearrangement also are indicated. On the right side of the scheme of each derivative, the chromosomes as seen in trypsin-Giemsa banding.
The latter was due to a deletion at band 11p11 of both der(11)s—namely, der(11)(11pter→11q23<11q23→11q25< 11q13→11qter) and der(11)(;11p13→11q13<22q11→ 22qter), followed by their fusion at this same band (Fig. 6). Moreover, the chromosome 11 painting probe revealed that both the mar1 and the mar2, without MLL signals, were derived from chromosome 11. These last markers, too, might have been formed by chromosome material from chromosome 11 short arms. In our patient, 111 and t(11;11;22) were accompanied by other karyotypic defects, and therefore we cannot establish if they were primary or secondary chromosome aberrations. In the literature, most 111 cases and one t(11;11) case were not discovered at disease presentation but at relapse. Our patient is uninformative on this point,
because he had already developed AML when he came to us for observation. Surely, he had undergone karyotype evolution because dic(11;11;22) and r(11) are further steps in comparison with der(11)(;11p13→11q13<22q11→ 22qter). The instability of his karyotype is shown by the presence of mar1 and mar2 in all the cells examined. We did not see, however, any unrelated clones [11]. Among the other abnormalities, monosomy for the long arm of chromosome 7 was seen in all the cells studied. It was due to an unbalanced t(7;17), defined only by FISH (Figs. 2c and 7). This technique, performed with the painting probe for chromosome 17, demonstrated that mar5 corresponded to der(17). An association between monosomy 7q and partial tandem duplication of the MLL gene has been suggested [16]. In fact, four cases with MLL du-
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Figure 6 Schematic representation of the suggested mechanism for the formation of dic(11;11;22)(22pter→ 22q11<11q13→11p11<11p11→ 11q23<11q23<11q23→11q25<11q13→11qter): the derivatives (11)(;11p13→ 11q13< 22q11→22qter) and (11)(11pter→11q23<11q23→ 11q25<11q13→11qter), are both deleted at band 11p11. Then the first one is translocated to the second one. The fusion occurs at band 11p11 of each derivative. On the right side of the figure, only the dic(11;11;22) is shown.
Figure 7 Schematic representation of normal chromosome 7, normal chromosome 17, der(7)(7pter→7q11<17q11→17qter), and der(17)(17pter→17q11;). On the right side of the scheme of each derivative, the chromosomes as seen in trypsin-Giemsa banding.
plication displayed loss of material from 7q. Even though we did not perform molecular analysis of the MLL gene, the studies cited may further suggest that, in our case, too, a partial tandem duplication of this gene might have occurred. REFERENCES 1. Harrison CJ, Cuneo A, Clark R, Johansson B, Lafage-Pochitaloff M, Mugneret F, Moorman AV, Secker-Walker LM (1998): Ten novel 11q23 chromosomal partner sites. Leukemia 12:811–822. 2. Thirman MJ, Gill HJ, Burnett RC, Mbangkollo D, McCabe NR, Kobayashi H, Ziemin-van der Poel S, Kaneko Y, Morgan R, Sandberg AA, Chaganti RSK, Larson RA, Le Beau MM, Diaz MO, Rowley JD (1993): Rearrangement of the MLL gene in acute lymphoblastic and acute myeloid leukemias with 11q23 chromosomal translocations. N Engl J Med 329:909–914. 3. Ziemin-van der Poel S, McCabe NR, Gill HJ, Espinosa R, Patel Y, Harden A, Rubineli P, Smith SD, Le Beau MM, Rowley JD, Diaz MO (1991): Identification of a gene, MLL, that spans the breakpoint in 11q23 translocations associated with human leukemias. Proc Natl Acad Sci USA 88:10735–10739. 4. Gu Y, Alder H, Nakamura T, Schichman SA, Prasad R, Canaani O, Saito H, Croce CM, Canaani E (1994): Sequence analysis of the breakpoint cluster region in the ALL-1 gene involved in acute leukemia. Cancer Res 54:2327–2330.
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5. Schichman SA, Caligiuri MA, Gu Y, Strout MP, Canaani E, Bloomfield CD, Croce CM (1994): ALL-1 partial duplication in acute leukemia. Proc Natl Acad Sci USA 91:6236–6239.
11 in chronic myelomonocytic leukemia: report of two cases and review of the literature. Cancer Genet Cytogenet 30:109– 117.
6. Rowley JD (1993): Rearrangements involving chromosome band 11q23 in acute leukemia. Sem Cancer Biol 4:377–385.
12. Ingram L, Raimondi SC, Mirro J, Rivera GK, Ragsdale ST, Behm F (1989): Characteristics of trisomy 11 in childhood acute leukemia with review of the literature. Leukemia 3:695–698.
7. Mrózek K, Heinonen K, Lawrence D, Carroll AJ, Koduru PRK, Rao KW, Strout MP, Hutchinson RE, Moore JO, Mayer RJ, Schiffer CA, Bloomfield CD (1997): Adult patients with de novo acute myeloid leukemia and t(9;11)(p22;q23) have a superior outcome to patients with other translocations involving band 11q23: a cancer and leukemia group B study. Blood 90:4532–4538. 8. Slovak ML, Traweek ST, Willman CL, Head DR, Kopecky KJ, Magenis RE, Appelbaum R, Forman SJ (1995): Trisomy 11: an association with stem/progenitor cell immunophenotype. B J Haematol 90:266–273. 9. ISCN (1995): Guidelines for Cancer Cytogenetics: Supplement to An International System for Human Cytogenetic Nomenclature. F Mitelman, ed. S. Karger, Basel. 10. Cuneo A, Bigoni R, Roberti MG, Bardi A, Rigolin GM, Piva N, Testoni N, Carboni C, Zaccaria A, Mecucci C, Matteucci C, La Starza R, Bernasconi P, Cavigliano P, Genini E, Mancini M, Nanni M, Alimena G, Castoldi GL (1998): Detection and monitoring of trisomy 8 by fluorescence in situ hybridization in acute myeloid leukemia: a multicentre study. Haematologica 83:21–26. 11. Takasaki N, Kaneko Y, Maseki N, Sakurai M (1988): Trisomy
13. Testa JR, Misawa S, Oguma N, Sloten KV, Wiernik PH (1985): Chromosome alterations in acute leukemia patients studied with improved culture methods. Cancer Res 45:430–434. 14. Leblanc T, Hillion J, Derrè J, Le Coniat M, Baruchel A, Daniel MT, Berger R (1994): Translocation t(11;11)(q13;q23) and HRX gene rearrangement associated with therapy-related leukemia in a child previously treated with VP16. Leukemia 8:1646–1648. 15. Cuneo A, Balboni M, Carli MG, Bigoni R, Roberti G, Pazzi I, Previati R, Castoldi GL (1994): Involvement of erythrocytic and granulomonocytic lineages by trisomy 11 in two cases of acute myelomonocytic leukemia with trilineage myelodysplasia: an interphase cytogenetic study. Cancer Genet Cytogenet 77:33–38. 16. Caligiuri MA, Strout MP, Schichman SA, Mrózek K, Arthur DC, Herzig GP, Baer M, Schiffer CA, Heinonen K, Knuutila S, Nousiainen T, Ruutu T, Block AMW, Shulman P, PedersenBjergaard J, Croce CM, Bloomfield CD (1996): Partial tandem duplication of ALL1 as a recurrent molecular defect in acute myeloid leukemia with trisomy 11. Cancer Res 56:1418–1425.
ABSTRACT: We describe a 73-year-old man diagnosed with acute myelomonocytic leukemia (AMLM4) following myelodysplasia with trisomy 11 and with a t(11;11;22) . This is the first case with both abnormalities present in the same cells and with the t(11;11;22) involving a chromosome 11 already duplicated at 11q23. This band contains the MLL gene that undergoes partial tandem duplication in patients with 111, which is “promiscuous,” being translocated with a large number of genetic partners. Our patient had a complex karyotype that was completely defined by in situ hybridization. This technique demonstrated that the t(11;11;22) derivative with a duplication of band 11q23 carried from three to four copies of MLL. Two copies of the gene were close to each other and centromeric to the breakpoint region. Therefore, a partial tandem duplication of the MLL gene might have happened before the occurrence of t(11;11;22). Considering the associated chromosome defects, the monosomy for the long arm of chromosome 7, due to an unbalanced translocation t(7;17), further underlines the possibility that a partial tandem duplication of the MLL gene might have taken place. © Elsevier Science Inc., 1999. All rights reserved. INTRODUCTION Recurrent chromosome abnormalities involving band 11q23 have been described in various hematologic disorders. This chromosome band can be joined to one of 25 other chromosomal regions and, recently, 10 further new partner chromosome sites were identified [1]. The majority of these translocations cause the rearrangement of the MLL gene, mapped at band 11q23 [2, 3]. Breakpoint clusters fall within exons 5 to 10 of the MLL gene [4]. All these translocations lead to the formation of a chimeric gene that is part MLL and part of a gene on the reciprocal chromosome. Recently, a different MLL rearrangement, consisting of partial tandem duplication of exons 2 through 6,
From the Istituto di Ematologia, Università di Pavia, Divisione di Ematologia, Policlinico San Matteo IRCCS, 27100 Pavia, Italy. Address reprint requests to: Dr. Paolo Bernasconi, Divisione di Ematologia, Policlinico San Matteo IRCCS, Piazzale Golgi N.5, 27100 Pavia, Italy. Received February 3, 1999; accepted May 25, 1999. Cancer Genet Cytogenet 116:111–118 (2000) Elsevier Science Inc., 1999. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
localized in the critical region of the fused MLL partner transcript, was reported in AML/MDS (acute myelomonocytic leukemia/myelodysplasia) patients with an isolated trisomy 11 (111) or with a normal karyotype [5]. Therefore, the fusion of a part of a putative proto-oncogene with itself was proposed as a novel mechanism for leukemogenesis. Clinically, 11q23 translocations or deletions are associated with myelomonocytic, monoblastic, or biphenotypic leukemia, showing a high blast cell count on presentation and a poor prognosis [6, 7], whereas 111 is associated with a stem/progenitor cell leukemia, having a poor response to standard chemotherapeutic regimens and a dismal prognosis [8]. We report a case of AML-M4 evolved from MDS, without previous exposure to topoisomerase II inhibitors, carrying a complex karyotype. This karyotype showed an unbalanced t(Y;5), leading to the almost complete loss of a chromosome 5 long arm and of a part of the Y chromosome between Ypter→Yq11, an unbalanced t(7;17), resulting in loss of a chromosome 7 long arm, trisomy 11, and a translocation t(11;11;22), leading to three derivatives.
0165-4608/00/$–see front matter PII S0165-4608(99)00117-X
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MATERIALS AND METHODS
CYTOGENETICS AND FISH RESULTS
Cytogenetic and Fluorescence In Situ Hybridization Cytogenetic analysis was carried out on bone marrow cells at diagnosis by using a trypsin-Giemsa banding technique. Metaphase cells were examined from short-term unstimulated cultures. Chromosomal abnormalities were defined according to ISCN [9]. Fluorescence in situ hybridization (FISH) was performed on cytogenetic preparations. At first we used three probes, all specific for chromosome 11: one centromeric probe, one painting probe, and one MLL (11q23) DNA probe. The last probe is specific for genomic sequences including the MLL locus. It detects the majority of rearrangements at the MLL gene locus by showing a large spot on the normal chromosome 11 and two smaller signals, one on the abnormal chromosome 11 and one at the site of the translocation. The 11q23 DNA probe does not detect translocations involving a 39 deletion of the MLL gene region. The centromeric probe was biotinylated (green spot); both of the other two were digoxigenated (red signals). Then, we employed digoxigenin-labelled painting probes for chromosomes 5, 17, and 22 (red signals). Two alphoid biotinylated probes for numbers 5 and 22 also were used (green spots). All these probes were commercially obtained (Oncor Inc., Gaithersburg, MD). Hybridization was carried out according to the manufacturer’s guidelines.
The karyotype was initially defined as: 47,X,2Y,del(5) (q13q33),der(7)(7pter→7q11<17q11→17qter), dup(11) (pter→ q23
CASE REPORT E. G., a 73-year-old man, never exposed to topoisomerase II inhibitors or to other genotoxic agents, came to us for observation because of fatigue and fever in November 1996. On physical examination, hepatomegaly (2 cm) was found. The patient’s blood count was as follows: hemoglobin, 8.1g/dl; white blood cell (WBC) count, 18.9 3 109/L (differential: neutrophils, 12%; lymphocytes, 8%; myelocytes, 12%; myeloblasts, 68%); and platelet count, 35.0 3 109/L. A bone marrow aspirate showed the almost exclusive presence (70%) of blasts, 22% with a monocytic appearance. Moreover, the morphologic features were suggestive of an underlying myelodysplasia because internuclear bridging in erythroid precursors, neutrophil hypogranulation, and micromegakaryocytes were noticed. Myeloperoxidase was positive in 88% of the blast cells, acetate esterase in 80%, and a-naphtyl acetate esterase, inhibited with NaF, in 42% of blast cells. The immunophenotyping of peripheral blood blast cells showed: HLA-DR (34%), CD34 (31%), CD33 (98%), CD13 (98%), CD14 (54%), CD15 (53%), CD11b (94%), and CD11c (89%). Serum lysozyme level was 82.3 mL/mL (normal value less than 10 mL/mL). The patient was classified as AML-M4, evolved from MDS. The chemotherapy regimen consisted of only 6-thioguanine, 40 mg in two daily doses for 5 days every 14 days, because of age and poor clinical conditions. After three cycles, there was a progressive rise in the WBC count and Ara-C, 80 mg/m2, in two daily doses for 5 days was started. The patient did not respond to this second treatment and died of sepsis.
Translocation (11;11;22) in a Case of MDS/AML
Figure 1 FISH definition of the t(11;11;22). (a) Hybridization with a chromosome 11 alphoid probe (green spots) on a cell with der(11)(;11p13→11q13<22q11→22qter)(mar3) (→) and with der(11)(11pter→11q23<11q23→ 11q25< 11q13→11qter) (----x): three copies of chromosome 11 are identified. (b) Hybridization with the MLL DNA probe (red signals) on the same metaphase, counterstained with DAPI: one spot is on the normal chromosome 11, three spots on der(11)(11pter→11q23<11q23→11q25< 11q13→11qter) (----x), none on the other derivative (mar3) (→). (c) Hybridization with chromosome 11 painting probe (red signals) on the same cell: the normal chromosome 11 and der(11)(11pter→ 11q23<11q23→11q25<11q13→11qter) (----x) are completely stained; der(11)(;11p13→ 11q13<22q11→ 22qter)(mar3) (→), mar1, and mar2 are only partly covered by the probe. (a9 through c9) The same hybridization steps on a cell with r(11) (.). (a0 through c0) The same hybridization steps on a cell with dic(11;11;22) (⇒). (c0) The dicentric is only partly stained by the painting probe. (d) Hybridization with a chromosome 22 painting probe (red signals) in a cell carrying der(11)(;11p13→11q13< 22q11→22qter) (→) and the other der(11) (----x) with only one centromere: one spot is on normal 22; the other one is on der(11)(;11p13→ 11q13<22q11→22qter) (→). (d9) Hybridization with the same probe on a cell with dic(11;11;22): one signal is on normal 22; the other one is on dic(11;11;22) (⇒). (e) FISH with chromosome 22 painting and alphoid probe (red and green spots) in a cell with dic(11;11;22) (⇒): one red signal is on the normal 22, and the other one is on dic(11;11;22) (⇒); one green spot is on the normal 22, and the other one identifies der(22)(mar4), consisting of only the centromere (c).
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Figure 2 FISH definition of the t(Y;5) and the t(7;17). (a) A DAPI-stained metaphase: the chromosome with the Y heterochromatic region is indicated (→). (a9) Hybridization with a 5a probe (green spots) on the preceding metaphase: one of the signals is on a chromosome with Y heterochromatic region (→), the other spot is on normal 5, and the last two are on chromosomes 1, inasmuch as the probe cross hybridizes with the centromere of this chromosome. (b) A DAPI-stained metaphase: the chromosome with Y heterochromatic region is indicated (→). (b9) Hybridization with a chromosome 5 and 22 painting probe (red signals) on the preceding metaphase: one signal is on a chromosome with Y heterochromatic region (→), and the largest one is on normal chromosome 5. The other spots identify normal 22 and dic(11;11;22) (⇒). (c) FISH with a chromosome 17 painting probe (red signals): three signals are observed, one corresponding to normal 17, one to der(7) (→), and one to mar5 (----x).
der(22)(22pter→22q11;),1der(?)t(?;11)(wcp111,cen112, MLL2),1der(?)t(?;11)(wcp111,cen112,MLL2)[5] (Fig. 4). DISCUSSION This is the first patient presenting with 111 and with a reciprocal t(11;11;22) in the same cells. Trisomy 11 has been reported in MDS, in AML, and in Philadelphia chromosome-negative chronic granulocytic leukemia [11, 12]. This numerical chromosome change is detected in about 1% of MDS/AML and does not correlate with any specific French–American–British subgroup of MDS/AML; probably, it marks a pluripotent stem cell disorder being discovered in primary as well as in secondary MDS/AML [8]. It has been suggested [11] that 111 is especially associated with myelomonocytic proliferation. The immunophenotyping of our patient is in line with that suggestion, because his blast cells expressed antigens of early myeloid progenitor cells (CD34, HLA-DR), of committed myeloid cells (CD15, CD13, CD33), and of monocytic cells (CD11b, CD11c). The expression of the first five antigens may be re-
lated to 111, whereas that of the last two may be related to the t(11;11). In fact, 11q23 translocations are typically associated with a M4-M5 cytotype. Moreover, the two t(11;11) cases reported in the literature [13, 14] also have been classified as M4 and M5b, respectively. The age range for 1 11 is from ,1 to 82 years, and a review of the literature indicates that from 30% to 50% of 111 patients have an underlying MDS, which frequently evolves into AML [8, 15]. A preleukemia phase was also present in a 13-year-old female with a t(11;11) and an MLL rearrangement [14]. Our patient, a 73-year-old man, probably had an unrecognized myelodysplasia before AML development because, at our observation, a trilineage dysplasia was still present in his marrow aspirate. Conventional cytogenetics allowed us to detect only the largest product of the t(11;11;22), which was interpreted as a dup(11). FISH, on the contrary, succeeded in completely defining the karyotype. At first, a chromosome 11 centromeric probe demonstrated that almost every cell (97%) carried 111. The successive use of the MLL probe showed this gene localized in a single copy on the normal chromo-
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Figure 3 Schematic representation of normal chromosomes 5 and Y and of der(5)(5pter→5q11
some 11 and in three copies on dup(11) with one centromere. Because the two red signals on the dup(11) resided close to each other and centromeric to the breakpoint regions, we suggest that this rearranged chro-
mosome 11 had had a duplication of band 11q23 before the occurrence of the t(11;11;22) (Fig. 5). This same chromosome carried a telomeric MLL signal, derived from the other product of the translocation der(11)(;11p13→ 11q13<22q11→22qter), which, in fact, did not show any spot due to MLL. Therefore, we suggest that the translocation took place between one chromosome 11 (already duplicated at band 11q23 and probably deleted within band 11q25), the other chromosome 11 (already deleted at 11p13, with a breakpoint at 11q13), and one chromosome 22 (deleted at band q11). The first der(11) received no material from the other two, the second der(11) obtained band 22q11→qter from chromosome 22, and the der(22) consisted of only the centromere. We do not know whether band 11q25 of der(11)(11pter→11q23<11q23→ 11q25<11q13→11qter) was lost; however, no material from chromosome 11 was present on the der(22) (Fig. 5). We did not study the MLL gene from a molecular point of view, and therefore we cannot establish whether this gene underwent a partial tandem duplication. Recently, it was reported that one attractive mechanism for this molecular defect involves a t(11;11)(q23;q23), occurring within the MLL gene locus [5]. In the present case, we cannot rule out the possibility that a breakpoint within band 11q23 might have happened, just when this same band underwent duplication (hypothesized step, Fig. 5). However, in our patient, genes residing within chromosome 11 short arm, too, might have been important for the development of his disease. In fact, the t(11;11;22) involved a chromosome 11 already deleted at p13. In five cells, we observed the absence of der(11)(;11p13→ 11q13<22q11→22qter) and the presence of dic(11;11;22).
Figure 4. Karyotype of a trypsin-Giemsa banded metaphase. Rearranged chromosomes are indicated by arrows.
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Figure 5 Schematic representation of the t(11;11;22). der(11)(;11p13→11q13<22q11→22qter)der(11)(11pter→ 11q23<11q23→11q25<11q13→11qter) and der(22)(22pter→22q11;) are shown. The hypothesized steps of the rearrangement also are indicated. On the right side of the scheme of each derivative, the chromosomes as seen in trypsin-Giemsa banding.
The latter was due to a deletion at band 11p11 of both der(11)s—namely, der(11)(11pter→11q23<11q23→11q25< 11q13→11qter) and der(11)(;11p13→11q13<22q11→ 22qter), followed by their fusion at this same band (Fig. 6). Moreover, the chromosome 11 painting probe revealed that both the mar1 and the mar2, without MLL signals, were derived from chromosome 11. These last markers, too, might have been formed by chromosome material from chromosome 11 short arms. In our patient, 111 and t(11;11;22) were accompanied by other karyotypic defects, and therefore we cannot establish if they were primary or secondary chromosome aberrations. In the literature, most 111 cases and one t(11;11) case were not discovered at disease presentation but at relapse. Our patient is uninformative on this point,
because he had already developed AML when he came to us for observation. Surely, he had undergone karyotype evolution because dic(11;11;22) and r(11) are further steps in comparison with der(11)(;11p13→11q13<22q11→ 22qter). The instability of his karyotype is shown by the presence of mar1 and mar2 in all the cells examined. We did not see, however, any unrelated clones [11]. Among the other abnormalities, monosomy for the long arm of chromosome 7 was seen in all the cells studied. It was due to an unbalanced t(7;17), defined only by FISH (Figs. 2c and 7). This technique, performed with the painting probe for chromosome 17, demonstrated that mar5 corresponded to der(17). An association between monosomy 7q and partial tandem duplication of the MLL gene has been suggested [16]. In fact, four cases with MLL du-
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Figure 6 Schematic representation of the suggested mechanism for the formation of dic(11;11;22)(22pter→ 22q11<11q13→11p11<11p11→ 11q23<11q23<11q23→11q25<11q13→11qter): the derivatives (11)(;11p13→ 11q13< 22q11→22qter) and (11)(11pter→11q23<11q23→ 11q25<11q13→11qter), are both deleted at band 11p11. Then the first one is translocated to the second one. The fusion occurs at band 11p11 of each derivative. On the right side of the figure, only the dic(11;11;22) is shown.
Figure 7 Schematic representation of normal chromosome 7, normal chromosome 17, der(7)(7pter→7q11<17q11→17qter), and der(17)(17pter→17q11;). On the right side of the scheme of each derivative, the chromosomes as seen in trypsin-Giemsa banding.
plication displayed loss of material from 7q. Even though we did not perform molecular analysis of the MLL gene, the studies cited may further suggest that, in our case, too, a partial tandem duplication of this gene might have occurred. REFERENCES 1. Harrison CJ, Cuneo A, Clark R, Johansson B, Lafage-Pochitaloff M, Mugneret F, Moorman AV, Secker-Walker LM (1998): Ten novel 11q23 chromosomal partner sites. Leukemia 12:811–822. 2. Thirman MJ, Gill HJ, Burnett RC, Mbangkollo D, McCabe NR, Kobayashi H, Ziemin-van der Poel S, Kaneko Y, Morgan R, Sandberg AA, Chaganti RSK, Larson RA, Le Beau MM, Diaz MO, Rowley JD (1993): Rearrangement of the MLL gene in acute lymphoblastic and acute myeloid leukemias with 11q23 chromosomal translocations. N Engl J Med 329:909–914. 3. Ziemin-van der Poel S, McCabe NR, Gill HJ, Espinosa R, Patel Y, Harden A, Rubineli P, Smith SD, Le Beau MM, Rowley JD, Diaz MO (1991): Identification of a gene, MLL, that spans the breakpoint in 11q23 translocations associated with human leukemias. Proc Natl Acad Sci USA 88:10735–10739. 4. Gu Y, Alder H, Nakamura T, Schichman SA, Prasad R, Canaani O, Saito H, Croce CM, Canaani E (1994): Sequence analysis of the breakpoint cluster region in the ALL-1 gene involved in acute leukemia. Cancer Res 54:2327–2330.
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