Molecular Cytogenetic Analysis Discloses Complex Genetic Imbalance in a t(11;21) Myelodysplastic Syndrome Simonetta Kerim, Giovanna Rege-Cambrin, Angelo Guerrasio, Claudia Rosso, and Herman Van Den Berghe
ABSTRACT: Three cases of t(11;21)(q24;q11.2) myelodysplastic syndromes (MDS) showed karyotypic evolution resulting in the presence of two der(11)t(11;211 without normal chromosome 11 and with partial trisomy 21q. In one of these, we performed further molecular cytogenetic investigations which showed 1) that this rearrangement led to changes in the dosage and location of both c-ets 1 and c-ets 2 protooncogenes; and 2) that the presence of two 11q+ chromosomes did not result from a nandisjunction, but that a second chromosome rearrangement had occurred. The final genetic imbalance resulting from this cytogenetic change involves at least hemizygosity for some sequences on the long arm of chromosome 11, including c-ets 1, plus trisomy for the most part of the long arm of chromosome 21, including c-ets 2. INTRODUCTION We recently described a new translocation It(11;21)(q24;q11.2)] occurring as a u n i q u e anomaly in myelodysplastic syndromes with rapid evolution [1]. The breakpoints of this t(11;21) are at or near the loci where the h u m a n homologue of v-ets sequences resides: c-ets 1 is assigned to 11q23-q25 [2], and c-ets 2 is mapped on chromosome 21 at band q22.1 [3]. Specific chromosomal abnormalities involving these regions have been associated with a wide range of hematological disorders [4-7], and several reports have demonstrated the relocation of c-ets 1 or c-ets 2 oncogenes as a consequence of translocations involving 11q23 or 21q22 breakpoints [8-11]. A most provocative finding in t(11;21) cells was the duplication, in all three patients, of the der(11)t(11;21) with disappearance of the normal 11. We originally interpreted this as resulting from n o n d i s j u n c t i o n resulting in homozygosity for the 11q+ marker, with s i m u l t a n e o u s loss of the normal 11. Because in our cases of t(11;21)(q24;q11.2) the breakpoint on chromosome 11 is apparently different from that in other hematological malignancies and both c-ets 1 and c-ets 2 are located on the chromosomes involved and because in addition two rearranged chromosomes 11q+ appeared to replace their normal counterparts, we decided to investigate the nature of this " d u p l i c a t i o n " and the fate of the c-ets genes further. From the Center for Human Genetics,Universityof Leuven,Belgium(S.K.,H.V.D.B.), and Dipartimento di ScienzeBiomedichee OncologiaUmana,SezioneClinica,Universityof Torino(G.R.-C.,A.G.,C.R.), Italy. Address reprint requests to H. Van Den Berghe, M.D., Center for Human Genetics, Herestraat 49, B-3000 Leuven, Belgium. Received August 24, 1989; accepted October 19, 1989.
243 © 1990 ElsevierSciencePublishingCo., Inc. 655 Avenueof the Americas,New York, NY 10010
Cancer Genet Cytogenet46:243-250 (1990) 0165-4608/90/$03.50
244
s. Kerim et al
MATERIALS AND METHODS Detailed clinical and cytogenetic information on the three patients with the t(11 ;21)(q24;q11.2) translocation was published previously [1]. In that study, Southern blotting and in situ hybridization studies were performed in only one case because we did not have fixed chromosome preparations and frozen DNA from leukemic ceils of the two other patients. In the present study, cells carrying t(11;21) were obtained from a patient with chronic myelomonocytic leukemia progressing to acute monoblastic leukemia. For cytogenetic analysis, bone marrow (BM) cells were cultured for 48 hours and mitoses were synchronized. Standard harvesting procedures were used, and chromosomes were G-banded with Wright stain. Cytogenetic nomenclature followed the InternatiopaJ System for Human Cytogenetic Nomenclature [12].
DNA Probes The human ets-1 probe was a 5.4-kilobase EcoRI fragment cloned into the EcoRI site of plasmid pKH 47 [2]. The Hu-ets 2 probe was a 1.023-kb EcoRI fragment [10]. flglobin was a 4.4-kb PstI genomic fragment [13], and Ha-Ras probe was a 6.4-kb BamHI genomic fragment [141.
Southern Blotting Analysis Bone marrow cells were obtained for DNA analysis in February 1987; at that time all metaphases showed t(11;21)(q24;q11.2) at cytogenetic investigation. After separation of BM cells on a Ficoll-Hypaque gradient, DNA was extracted by the phenohchlorophorm technique, digested with several restriction enzymes, run on agarose gel, and transferred onto a nitrocellulose paper as previously described [15]. The resulting fragments were hybridized with Hu-ets 1,/3-globin, and Ha-ras probes. Hybridization was performed independently on the top and bottom pellets, enriched in blasts and mature cells, respectively.
In situ Chromosomal Hybridization The c-ets 1 and c-ets 2 probes were nick-translated using 3H-nucleotides to a specific activity of about 1-2 × 107 dpm//~g and hybridized in situ to metaphase cells with t(11;211, following the technique described by Harper and Saunders [16]. Normal metaphases from peripheral blood phytohemagglutinin (PHA)-stimulated cells were also hybridized with the same probes to serve as a control. Final probe concentration was 100-200 ng/ml hybridization mixture (50% formamide, 2 × SSC, 10% dextran sulfate, and salmon sperm DNA). After hybridization the slides were coated with Kodak NTB-2 emulsion and kept in the dark at 4°C for 7-21 days. After development, chromosomes were stained with Wright solution and photographed. Labeling significance was checked by chi-square analysis, testing the hypothesis that labeling is random over all chromosomes.
RESULTS Chromosome Analysis t(11;21)(q24;q11.2) with duplication of the marker chromosome 11q+ and loss of the normal homologue characterized all metaphases in BM culture. No other chromosomel abnormalities were detected.
t(11;21) M y e l o d y s p l a s t i c S y n d r o m e
245
Fate of e t s Genes In situ h y b r i d i z a t i o n analysis. In the h y b r i d i z a t i o n study with the c-ets 1 probe there was no h y b r i d i z a t i o n to the marker c h r o m o s o m e 11q +; a cluster of grains was observed on the c h r o m o s o m e 2 1 q - instead. The grains in this cluster represented 10.2% of all grains observed over all c h r o m o s o m e s (18 of 178: p ~ 0.0001 by chisquare test), indicating that the c-ets 1 gene had been translocated from c h r o m o s o m e 11 to the 2 1 q - (Fig. 1). Hybridization of t(11;21) metaphases with the h u m a n c-ets 2 probe showed only one grain on the 2 1 q - c h r o m o s o m e whereas the 11q+ chromosomes contained eight grains (of 96; p ~ 0.0005), w h i c h were clustered at the junction of the translocation breakpoint. These results are consistent with the relocation of the c-ets 2 gene at the 11q + chromosomes. S o u t h e r n blotting studies. Southern analysis of DNA from leukemic cells after BamHI and HindlII digestion and h y b r i d i z a t i o n with the Hu-ets 1 probe showed only a germline fragment, indicating that c-ets 1 was not rearranged in leukemic cells. The intensity of the band in the patient's DNA, however, was a p p r o x i m a t e l y half as c o m p a r e d with that of control DNA, which is very suggestive for the loss of one Huets 1 allele in the leukemic cells (Fig. 2).
Nature of the Duplicated 11q+ Because a normal c h r o m o s o m e 11 was not present and the 11q + c h r o m o s o m e was a p p a r e n t l y duplicated, homozygosity of all genes located on the rearranged chromosome 11 was expected if d u p l i c a t i o n was the result of nondisjunction. To verify this hypothesis, we analyzed leukemic DNA samples from our patient by blot hybridization with m o l e c u l a r probes that detect p o l y m o r p h i s m s on the short arm of the chromosome 11. The informative markers used were the/3-globin gene, localized at 11p15 [17] and the Ha-ras oncogene assigned to 11p15.1-p15.5 [18]. Hybridization studies with ~-globin and Ha-ras probes, after digestion with AvaII restriction enzyme, showed the presence of the two allelic fragments with both probes (Fig. 3): two Haras alleles were also observed in BamHI digestion. Two p o l y m o r p h i c bands of the same intensity were present in the DNA derived from either the top and bottom cell fractions, thus excluding a contamination of normal cells, w h i c h could have gone u n d e t e c t e d in the cytogenetic analysis. The data suggest that sequences belonging to the two different chromosomes 11 were present and that the second 11q + chromosome did not originate from a d u p l i c a t i o n of the first.
DISCUSSION The above-described results reveal some interesting aspects of this case of t(11;21). First, in situ h y b r i d i z a t i o n analysis performed on t(11;21) metaphases proved that this cytogenetic change leads to relocation of both c-ets 1 and c-ets 2 on 2 1 q - and 11q + chromosomes, respectively. The transposition of c-ets 1 is the consequence of a breakpoint at band 11q24, different from the 11q23 band, w h i c h is much more often involved in so-called characteristic c h r o m o s o m e changes in leukemia, e.g., t(9;11)(p21;q23) [5]. In these cases ets 1 as a rule is translocated [8-11[. Our finding may thus lead to a more precise localization of this oncogene below the 11q23 band, at 11q24-q25. Furthermore, Southern blot analysis of p o l y m o r p h i c genes located on the short arm of c h r o m o s o m e 11 showed that our previous interpretation of the karyotype, n a m e l y loss of the normal 11 and d u p l i c a t i o n of the 11q + chromosome, was incorrect.
246
s. Kerim et al
ETS I
P p "q
P 11.2 ~ . O O O O O O , , . . . . . O O H .
21qq24
11q,
ETS 2
11.2
Cl
•
21q00000000
q24
11q, Figure 1 Distribution of labeled sites on t(11;21) metaphases after hybridization with Hu-ets 1 and Hu-ets 2 probes.
The second and seemingly identical 11q + may have arisen from two different mitotic mechanisms: as a result of an exchange between a previously duplicated 11q + and the normal 11 or, alternatively, from a second t(11;21) after occurrence of trisomy 21, with subsequent loss of the 2 1 q - resulting from this second translocation. Which of these two events occurred could not be determined; adequate probes for testing whether some sequences between the polymorphic Haoras and fl-globin genes and the 11q24 region underwent homozygosity were not available to us. Whatever the mechanism, the cell(s) carrying two 11q + chromosomes without a normal 11 appear to have acquired a proliferative advantage. This is particularly clear in the two other described patients in w h o m only one t(11;21) was found initially but in w h o m cells with two t(11;21) gradually dominated as their disease progressed [1].
24 7
t(11;21) Myelodys plastic S y n d r o m e
ECO
RI
-5,4 Kb
1
2
3
E T S - 1 probe Figure 2 Hybridization with the Hu-ets 1 probe after Eco-RI digestion. Control DNA (lane 1), patient's DNA from bone marrow (BM) cells of the top pellet after Ficoll separation (lane 2); patient's DNA from BM cells of the bottom pellet (lane 3).
This proliferative advantage may result at least in part from the occurrence of further genetic changes as a consequence of the 11q÷ " d u p l i c a t i o n " and loss of the normal 11. Such genetic changes did occur in this case. First, one e t s 1 allele was lost. Southern blot analysis i n d i c a t e d that the activity of e t s 1 was only about half of that of normal controls. Loss of one e t s 1 allele belonging to the normal 11 may thus have occurred after formation of the second 11q ÷ . The function of the e t s 1 protooncogene is unknown. Deletion of one e t s 1 copy has been demonstrated in myelogenous cell lines with c h r o m o s o m e change at 11q24 [19] and deletions of the type del(11)(q23), in w h i c h the c - e t s 1 will be lost, are often observed in hematological malignancies, especially of m y e l o m o n o c y t i c lineage [6]. Therefore, it is t e m p t i n g to speculate that the dosage of certain genes (including 1) located in this region may have a role in
248
s. Kerim et al
C
T ABVa II enzYmre B Kb
Kb
--2.2 _
2.0
--1.9
_1.1
B E T A GLOBIN
A
HAoRAS
B
Figure 3 Digestion with Avail enzyme and hybridization with two probes located on the short arm of chromosome 11:/J-globin and Ha-ras. Control DNA (lane C); patient's DNA from bone marrow (BM} cells of the top pellet after Ficoll separation, enriched in immature cells (lane T); patient's DNA from BM cells of the bottom pellet containing mature myeloid cells (lane B}. In the BM cells, a polymorphism is present for both fl-globin and Ha-ras genes.
control of cellular proliferation and differentiation. The finding of low levels of c-ets 1-related mRNA in leukemic cells with t(4;11) 110] might support this hypothesis; the amplification of ets 1 after chromosome change involving 11q23 in two patients with acute myelomonocytic leukemia and l y m p h o m a [9], however, may not support it. Second, there was trisomy of genes located at 21q11.2 to 21qter. The high prevalence of trisomy 21 in acute myelogenous leukemias and myelodysplastic syndromes [6] suggests a role in proliferation for the dosage of genes belonging to this chromosome. In our case, segment 21q11.2 to 21qter may thus carry genes whose duplication is important in neoplastic proliferation. Among these genes is the ets 2 protooncogene.
t(11;21) Myelodysplastic S y n d r o m e
249
Evidence exists of its altered expression after c h r o m o s o m e change with i n v o l v e m e n t of 21q22 I101, but w h e th e r this gene has any role in the pathogenesis of malignancy is unknown. In summary, this molecular cytogenetic study on the newly described t(11;21) in MDS has s h o w n that c-ets 1 normally located on 11q and c-ets 2 normally located on 21q are both translocated in this c h r o m o s o m e rearrangement, and c-ets 1 is relocated not only in cases with rearrangement on c h r o m o s o m e 11 involving band q23 but in this case also in v o lv in g band q24. In the course of the disease, a second ch r o m o so m al rearrangement leading to loss of the normal 11 and to production of a seemingly duplicated 11q + occurred, possibly consisting of an exchange between a previously duplicated 11q+ and the normal 11, s o m e w h e r e between the p o l y m o r p h i c Ha-ras and/3-globin genes and the 11q24 region, or as the product of a new t(11:21). This observation shows that duplication of a rearranged chromosotne and concomitant loss of its normal counterpart, often observed in malignant cells, may not necessarily involve simple non-disjunction m e c h a n i s m s as is generally assumed. The final genetic imbalance resulting from all these rearrangements must be complex, as it may involve hemizigosity m i n i m a l l y for some sequences of 11q, i n cl u d i n g c-ets 1, and trisomy of most of the long arm of c h r o m o s o m e 21, i n cl u d i n g c-ets 2. We do not know w h et h er the same genetic imbalance also occurred in the two other cases with t(11;21) but, because of the great similarity between all three patients, it may well have. Supported by the Inter-university Network for Fundamental Research sponsored by the Belgian Government (1987-1991), in part by a grant from AIRC to G.S., and by Grant No. 8800784.44 from CNR, Progetto Finalizzato Oncologia. S.K. is a fellow of the Associazione Italiana per la Ricerca sul Cancro. The authors wish to thank Drs. Dominique Stehelin and Nicoletta Sacchi for providing c-ets 1 and 2 probes, respectively.
REFERENCES 1. Rege Cambrin G, Mecucci C, Kerim S, Scaravaglio P, Boogaerts M, Van Den Berghe H (1988): Translocation t(11;21))q24;qll.2) is a new nonrandomly occurring chromosome change in myelodysplastic syndromes. Cancer Genet Cytogenet 34:33-40. 2. De Taisne C, Gregonne A, Stehelin D, Bernheim A, Berger R (1984): Chromosomal localization of the human protooncogene c-ets. Nature 310:581-583. 3. Watson DK, McWilliams-Smith MJ, Kozak C, Reeves R, Gearhart J, Nunn MF, Nash W, Fowle JR, Duesberg P, Papas TS, O'Brien SJ (1986): Conserved chromosomal position of dual domain of the ets protooncogene in cats, mice and humans. Proc Natl Acad Sci USA 83:1792-1796. 4. Van Den Berghe H, David G, Broeckaert-Van Orshoven A, Louwagie A. Verwilghen R, Casteels-Van Daele H, Eggemont E, Eeckels R (1979): A new chromosome anomaly in acute lymphoblastic leukemia. Hum Genet 46:173-180. 5. Hagemeijer A, Hahlen K, Sizoo W, Abels J (1982): Translocation (9;11)(p21q23) in three cases of acute monoblastic leukemia. Cancer Genet Cytogenet 5:95-105. 6. Fourth International Workshop on Chromosomes in Leukemia: A prospective study of acute nonlymphocytic leukemia (1984): Cancer Genet Cytogenet 11:294-360. 7. Tricot G, Broeckaert-Van Orshoven A, Casteels-Van Daele M, Van Den Berghe H (1981): 8/ 21 Translocation in acute myeloid leukemia. Scand J Haematol 26:168-176. 8. Diaz MO, Le Beau MM, Pitha P, Rowley JD (1986): Interferon and c-ets genes in the translocation (9;11)(p22q23) in human acute monocytic leukemia. Science 231:265-267. 9. Rovigatti U, Watson DK, Yunis JJ (1986): Amplification and rearrangement of Hu-ets-1 in leukemia and lymphoma with involvement of 11q23. Science 232:398-400. 10. Sacchi H, Watson DK, Geurts Van Kessel Ad HM, Hagemeijer A, Kersey J, Drabkin HD, Patterson D, Papas TS (1986): Hu-ets-1 and Hu-ets-2 genes are transposed in acute leukemias with (4;11) and (8;21) translocations. Science 231:379-381.
250
s. K e r i m et al
11. Morris CM, Whitham SE, Fitzgerald PH (1988): Transposition of the oncogene c-ets-1 in a t(11;19)(q23;p13) cell line transient during clonal evolution of blast crisis chronic myeloid leukemia. Leukemia 2:74-78. 12. ISCN (1985): An International System for Human Cytogenetic Nomenclature, Harnden DG, Klinger HP (eds); published in collaboration with Cytogenet Cell Genet (Karger, Basel, 1985); also in Birth Defects: Original Article Series, Vol. 21, No. 1 (March of Dimes Birth Defects Foundation, New York, 1985). 13. Camaschella C, Saglio G. Serra A, Sampietro M, Cappellini MD, Fiorelli G, Cremonesi L, Tedeschi S, Ferrari M (1985): DNA Polymorphism Analysis in the Italian Population and Prenatal Diagnosis of Thalassemia in First Trimester Fetal Diagnosis, M Fraccaro, G Simoni, B Brambati, eds. Springer-Verlag, Berlin. 14. Saglio G, Camaschella C, Giai M, Serra A, Guerrasio A, Peirone B, Gasparini P, Mazza U, Ceppellini R, Biglia N, Cortese P, Sismondi P (1988): Distribution of Ha-ras-1 protooncogene alleles in breast cancer patients and in control population. Breast Cancer Res Treat 11:147-153. 15. Maniatis T, Fritsch EF, Sambrook J (1982): Molecular Cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 16. Harper ME, Saunders GF (1981): Localization of single copy DNA sequences on G-banded h u m a n chromosomes by in sity hybridization. Chromosoma 83:431-439. 17. de Martinville B, Francke U (1983): The H-ras 1 insulin and ~-globin loci map outside the deletion associated with aniridia-Wilms' tumor. Nature 305:641-643. 18. de Martinville B, Giacalone J (1983): Oncogene from h u m a n EJ bladder carcinoma is located on the short arm of chromosome 11. Science 219:498-503. 19. Ohyashiki K, Ohyashiki JH, Sandberg AA, Minowada J, Kinniburg AJ (1987): Loss of a Huets-a allele in h u m a n leukemia cell lines ML-1, -2, and 3 with a chromosome change at 11q24. Cancer Res 47:3842-3846.
ABSTRACT: Three cases of t(11;21)(q24;q11.2) myelodysplastic syndromes (MDS) showed karyotypic evolution resulting in the presence of two der(11)t(11;211 without normal chromosome 11 and with partial trisomy 21q. In one of these, we performed further molecular cytogenetic investigations which showed 1) that this rearrangement led to changes in the dosage and location of both c-ets 1 and c-ets 2 protooncogenes; and 2) that the presence of two 11q+ chromosomes did not result from a nandisjunction, but that a second chromosome rearrangement had occurred. The final genetic imbalance resulting from this cytogenetic change involves at least hemizygosity for some sequences on the long arm of chromosome 11, including c-ets 1, plus trisomy for the most part of the long arm of chromosome 21, including c-ets 2. INTRODUCTION We recently described a new translocation It(11;21)(q24;q11.2)] occurring as a u n i q u e anomaly in myelodysplastic syndromes with rapid evolution [1]. The breakpoints of this t(11;21) are at or near the loci where the h u m a n homologue of v-ets sequences resides: c-ets 1 is assigned to 11q23-q25 [2], and c-ets 2 is mapped on chromosome 21 at band q22.1 [3]. Specific chromosomal abnormalities involving these regions have been associated with a wide range of hematological disorders [4-7], and several reports have demonstrated the relocation of c-ets 1 or c-ets 2 oncogenes as a consequence of translocations involving 11q23 or 21q22 breakpoints [8-11]. A most provocative finding in t(11;21) cells was the duplication, in all three patients, of the der(11)t(11;21) with disappearance of the normal 11. We originally interpreted this as resulting from n o n d i s j u n c t i o n resulting in homozygosity for the 11q+ marker, with s i m u l t a n e o u s loss of the normal 11. Because in our cases of t(11;21)(q24;q11.2) the breakpoint on chromosome 11 is apparently different from that in other hematological malignancies and both c-ets 1 and c-ets 2 are located on the chromosomes involved and because in addition two rearranged chromosomes 11q+ appeared to replace their normal counterparts, we decided to investigate the nature of this " d u p l i c a t i o n " and the fate of the c-ets genes further. From the Center for Human Genetics,Universityof Leuven,Belgium(S.K.,H.V.D.B.), and Dipartimento di ScienzeBiomedichee OncologiaUmana,SezioneClinica,Universityof Torino(G.R.-C.,A.G.,C.R.), Italy. Address reprint requests to H. Van Den Berghe, M.D., Center for Human Genetics, Herestraat 49, B-3000 Leuven, Belgium. Received August 24, 1989; accepted October 19, 1989.
243 © 1990 ElsevierSciencePublishingCo., Inc. 655 Avenueof the Americas,New York, NY 10010
Cancer Genet Cytogenet46:243-250 (1990) 0165-4608/90/$03.50
244
s. Kerim et al
MATERIALS AND METHODS Detailed clinical and cytogenetic information on the three patients with the t(11 ;21)(q24;q11.2) translocation was published previously [1]. In that study, Southern blotting and in situ hybridization studies were performed in only one case because we did not have fixed chromosome preparations and frozen DNA from leukemic ceils of the two other patients. In the present study, cells carrying t(11;21) were obtained from a patient with chronic myelomonocytic leukemia progressing to acute monoblastic leukemia. For cytogenetic analysis, bone marrow (BM) cells were cultured for 48 hours and mitoses were synchronized. Standard harvesting procedures were used, and chromosomes were G-banded with Wright stain. Cytogenetic nomenclature followed the InternatiopaJ System for Human Cytogenetic Nomenclature [12].
DNA Probes The human ets-1 probe was a 5.4-kilobase EcoRI fragment cloned into the EcoRI site of plasmid pKH 47 [2]. The Hu-ets 2 probe was a 1.023-kb EcoRI fragment [10]. flglobin was a 4.4-kb PstI genomic fragment [13], and Ha-Ras probe was a 6.4-kb BamHI genomic fragment [141.
Southern Blotting Analysis Bone marrow cells were obtained for DNA analysis in February 1987; at that time all metaphases showed t(11;21)(q24;q11.2) at cytogenetic investigation. After separation of BM cells on a Ficoll-Hypaque gradient, DNA was extracted by the phenohchlorophorm technique, digested with several restriction enzymes, run on agarose gel, and transferred onto a nitrocellulose paper as previously described [15]. The resulting fragments were hybridized with Hu-ets 1,/3-globin, and Ha-ras probes. Hybridization was performed independently on the top and bottom pellets, enriched in blasts and mature cells, respectively.
In situ Chromosomal Hybridization The c-ets 1 and c-ets 2 probes were nick-translated using 3H-nucleotides to a specific activity of about 1-2 × 107 dpm//~g and hybridized in situ to metaphase cells with t(11;211, following the technique described by Harper and Saunders [16]. Normal metaphases from peripheral blood phytohemagglutinin (PHA)-stimulated cells were also hybridized with the same probes to serve as a control. Final probe concentration was 100-200 ng/ml hybridization mixture (50% formamide, 2 × SSC, 10% dextran sulfate, and salmon sperm DNA). After hybridization the slides were coated with Kodak NTB-2 emulsion and kept in the dark at 4°C for 7-21 days. After development, chromosomes were stained with Wright solution and photographed. Labeling significance was checked by chi-square analysis, testing the hypothesis that labeling is random over all chromosomes.
RESULTS Chromosome Analysis t(11;21)(q24;q11.2) with duplication of the marker chromosome 11q+ and loss of the normal homologue characterized all metaphases in BM culture. No other chromosomel abnormalities were detected.
t(11;21) M y e l o d y s p l a s t i c S y n d r o m e
245
Fate of e t s Genes In situ h y b r i d i z a t i o n analysis. In the h y b r i d i z a t i o n study with the c-ets 1 probe there was no h y b r i d i z a t i o n to the marker c h r o m o s o m e 11q +; a cluster of grains was observed on the c h r o m o s o m e 2 1 q - instead. The grains in this cluster represented 10.2% of all grains observed over all c h r o m o s o m e s (18 of 178: p ~ 0.0001 by chisquare test), indicating that the c-ets 1 gene had been translocated from c h r o m o s o m e 11 to the 2 1 q - (Fig. 1). Hybridization of t(11;21) metaphases with the h u m a n c-ets 2 probe showed only one grain on the 2 1 q - c h r o m o s o m e whereas the 11q+ chromosomes contained eight grains (of 96; p ~ 0.0005), w h i c h were clustered at the junction of the translocation breakpoint. These results are consistent with the relocation of the c-ets 2 gene at the 11q + chromosomes. S o u t h e r n blotting studies. Southern analysis of DNA from leukemic cells after BamHI and HindlII digestion and h y b r i d i z a t i o n with the Hu-ets 1 probe showed only a germline fragment, indicating that c-ets 1 was not rearranged in leukemic cells. The intensity of the band in the patient's DNA, however, was a p p r o x i m a t e l y half as c o m p a r e d with that of control DNA, which is very suggestive for the loss of one Huets 1 allele in the leukemic cells (Fig. 2).
Nature of the Duplicated 11q+ Because a normal c h r o m o s o m e 11 was not present and the 11q + c h r o m o s o m e was a p p a r e n t l y duplicated, homozygosity of all genes located on the rearranged chromosome 11 was expected if d u p l i c a t i o n was the result of nondisjunction. To verify this hypothesis, we analyzed leukemic DNA samples from our patient by blot hybridization with m o l e c u l a r probes that detect p o l y m o r p h i s m s on the short arm of the chromosome 11. The informative markers used were the/3-globin gene, localized at 11p15 [17] and the Ha-ras oncogene assigned to 11p15.1-p15.5 [18]. Hybridization studies with ~-globin and Ha-ras probes, after digestion with AvaII restriction enzyme, showed the presence of the two allelic fragments with both probes (Fig. 3): two Haras alleles were also observed in BamHI digestion. Two p o l y m o r p h i c bands of the same intensity were present in the DNA derived from either the top and bottom cell fractions, thus excluding a contamination of normal cells, w h i c h could have gone u n d e t e c t e d in the cytogenetic analysis. The data suggest that sequences belonging to the two different chromosomes 11 were present and that the second 11q + chromosome did not originate from a d u p l i c a t i o n of the first.
DISCUSSION The above-described results reveal some interesting aspects of this case of t(11;21). First, in situ h y b r i d i z a t i o n analysis performed on t(11;21) metaphases proved that this cytogenetic change leads to relocation of both c-ets 1 and c-ets 2 on 2 1 q - and 11q + chromosomes, respectively. The transposition of c-ets 1 is the consequence of a breakpoint at band 11q24, different from the 11q23 band, w h i c h is much more often involved in so-called characteristic c h r o m o s o m e changes in leukemia, e.g., t(9;11)(p21;q23) [5]. In these cases ets 1 as a rule is translocated [8-11[. Our finding may thus lead to a more precise localization of this oncogene below the 11q23 band, at 11q24-q25. Furthermore, Southern blot analysis of p o l y m o r p h i c genes located on the short arm of c h r o m o s o m e 11 showed that our previous interpretation of the karyotype, n a m e l y loss of the normal 11 and d u p l i c a t i o n of the 11q + chromosome, was incorrect.
246
s. Kerim et al
ETS I
P p "q
P 11.2 ~ . O O O O O O , , . . . . . O O H .
21qq24
11q,
ETS 2
11.2
Cl
•
21q00000000
q24
11q, Figure 1 Distribution of labeled sites on t(11;21) metaphases after hybridization with Hu-ets 1 and Hu-ets 2 probes.
The second and seemingly identical 11q + may have arisen from two different mitotic mechanisms: as a result of an exchange between a previously duplicated 11q + and the normal 11 or, alternatively, from a second t(11;21) after occurrence of trisomy 21, with subsequent loss of the 2 1 q - resulting from this second translocation. Which of these two events occurred could not be determined; adequate probes for testing whether some sequences between the polymorphic Haoras and fl-globin genes and the 11q24 region underwent homozygosity were not available to us. Whatever the mechanism, the cell(s) carrying two 11q + chromosomes without a normal 11 appear to have acquired a proliferative advantage. This is particularly clear in the two other described patients in w h o m only one t(11;21) was found initially but in w h o m cells with two t(11;21) gradually dominated as their disease progressed [1].
24 7
t(11;21) Myelodys plastic S y n d r o m e
ECO
RI
-5,4 Kb
1
2
3
E T S - 1 probe Figure 2 Hybridization with the Hu-ets 1 probe after Eco-RI digestion. Control DNA (lane 1), patient's DNA from bone marrow (BM) cells of the top pellet after Ficoll separation (lane 2); patient's DNA from BM cells of the bottom pellet (lane 3).
This proliferative advantage may result at least in part from the occurrence of further genetic changes as a consequence of the 11q÷ " d u p l i c a t i o n " and loss of the normal 11. Such genetic changes did occur in this case. First, one e t s 1 allele was lost. Southern blot analysis i n d i c a t e d that the activity of e t s 1 was only about half of that of normal controls. Loss of one e t s 1 allele belonging to the normal 11 may thus have occurred after formation of the second 11q ÷ . The function of the e t s 1 protooncogene is unknown. Deletion of one e t s 1 copy has been demonstrated in myelogenous cell lines with c h r o m o s o m e change at 11q24 [19] and deletions of the type del(11)(q23), in w h i c h the c - e t s 1 will be lost, are often observed in hematological malignancies, especially of m y e l o m o n o c y t i c lineage [6]. Therefore, it is t e m p t i n g to speculate that the dosage of certain genes (including 1) located in this region may have a role in
248
s. Kerim et al
C
T ABVa II enzYmre B Kb
Kb
--2.2 _
2.0
--1.9
_1.1
B E T A GLOBIN
A
HAoRAS
B
Figure 3 Digestion with Avail enzyme and hybridization with two probes located on the short arm of chromosome 11:/J-globin and Ha-ras. Control DNA (lane C); patient's DNA from bone marrow (BM} cells of the top pellet after Ficoll separation, enriched in immature cells (lane T); patient's DNA from BM cells of the bottom pellet containing mature myeloid cells (lane B}. In the BM cells, a polymorphism is present for both fl-globin and Ha-ras genes.
control of cellular proliferation and differentiation. The finding of low levels of c-ets 1-related mRNA in leukemic cells with t(4;11) 110] might support this hypothesis; the amplification of ets 1 after chromosome change involving 11q23 in two patients with acute myelomonocytic leukemia and l y m p h o m a [9], however, may not support it. Second, there was trisomy of genes located at 21q11.2 to 21qter. The high prevalence of trisomy 21 in acute myelogenous leukemias and myelodysplastic syndromes [6] suggests a role in proliferation for the dosage of genes belonging to this chromosome. In our case, segment 21q11.2 to 21qter may thus carry genes whose duplication is important in neoplastic proliferation. Among these genes is the ets 2 protooncogene.
t(11;21) Myelodysplastic S y n d r o m e
249
Evidence exists of its altered expression after c h r o m o s o m e change with i n v o l v e m e n t of 21q22 I101, but w h e th e r this gene has any role in the pathogenesis of malignancy is unknown. In summary, this molecular cytogenetic study on the newly described t(11;21) in MDS has s h o w n that c-ets 1 normally located on 11q and c-ets 2 normally located on 21q are both translocated in this c h r o m o s o m e rearrangement, and c-ets 1 is relocated not only in cases with rearrangement on c h r o m o s o m e 11 involving band q23 but in this case also in v o lv in g band q24. In the course of the disease, a second ch r o m o so m al rearrangement leading to loss of the normal 11 and to production of a seemingly duplicated 11q + occurred, possibly consisting of an exchange between a previously duplicated 11q+ and the normal 11, s o m e w h e r e between the p o l y m o r p h i c Ha-ras and/3-globin genes and the 11q24 region, or as the product of a new t(11:21). This observation shows that duplication of a rearranged chromosotne and concomitant loss of its normal counterpart, often observed in malignant cells, may not necessarily involve simple non-disjunction m e c h a n i s m s as is generally assumed. The final genetic imbalance resulting from all these rearrangements must be complex, as it may involve hemizigosity m i n i m a l l y for some sequences of 11q, i n cl u d i n g c-ets 1, and trisomy of most of the long arm of c h r o m o s o m e 21, i n cl u d i n g c-ets 2. We do not know w h et h er the same genetic imbalance also occurred in the two other cases with t(11;21) but, because of the great similarity between all three patients, it may well have. Supported by the Inter-university Network for Fundamental Research sponsored by the Belgian Government (1987-1991), in part by a grant from AIRC to G.S., and by Grant No. 8800784.44 from CNR, Progetto Finalizzato Oncologia. S.K. is a fellow of the Associazione Italiana per la Ricerca sul Cancro. The authors wish to thank Drs. Dominique Stehelin and Nicoletta Sacchi for providing c-ets 1 and 2 probes, respectively.
REFERENCES 1. Rege Cambrin G, Mecucci C, Kerim S, Scaravaglio P, Boogaerts M, Van Den Berghe H (1988): Translocation t(11;21))q24;qll.2) is a new nonrandomly occurring chromosome change in myelodysplastic syndromes. Cancer Genet Cytogenet 34:33-40. 2. De Taisne C, Gregonne A, Stehelin D, Bernheim A, Berger R (1984): Chromosomal localization of the human protooncogene c-ets. Nature 310:581-583. 3. Watson DK, McWilliams-Smith MJ, Kozak C, Reeves R, Gearhart J, Nunn MF, Nash W, Fowle JR, Duesberg P, Papas TS, O'Brien SJ (1986): Conserved chromosomal position of dual domain of the ets protooncogene in cats, mice and humans. Proc Natl Acad Sci USA 83:1792-1796. 4. Van Den Berghe H, David G, Broeckaert-Van Orshoven A, Louwagie A. Verwilghen R, Casteels-Van Daele H, Eggemont E, Eeckels R (1979): A new chromosome anomaly in acute lymphoblastic leukemia. Hum Genet 46:173-180. 5. Hagemeijer A, Hahlen K, Sizoo W, Abels J (1982): Translocation (9;11)(p21q23) in three cases of acute monoblastic leukemia. Cancer Genet Cytogenet 5:95-105. 6. Fourth International Workshop on Chromosomes in Leukemia: A prospective study of acute nonlymphocytic leukemia (1984): Cancer Genet Cytogenet 11:294-360. 7. Tricot G, Broeckaert-Van Orshoven A, Casteels-Van Daele M, Van Den Berghe H (1981): 8/ 21 Translocation in acute myeloid leukemia. Scand J Haematol 26:168-176. 8. Diaz MO, Le Beau MM, Pitha P, Rowley JD (1986): Interferon and c-ets genes in the translocation (9;11)(p22q23) in human acute monocytic leukemia. Science 231:265-267. 9. Rovigatti U, Watson DK, Yunis JJ (1986): Amplification and rearrangement of Hu-ets-1 in leukemia and lymphoma with involvement of 11q23. Science 232:398-400. 10. Sacchi H, Watson DK, Geurts Van Kessel Ad HM, Hagemeijer A, Kersey J, Drabkin HD, Patterson D, Papas TS (1986): Hu-ets-1 and Hu-ets-2 genes are transposed in acute leukemias with (4;11) and (8;21) translocations. Science 231:379-381.
250
s. K e r i m et al
11. Morris CM, Whitham SE, Fitzgerald PH (1988): Transposition of the oncogene c-ets-1 in a t(11;19)(q23;p13) cell line transient during clonal evolution of blast crisis chronic myeloid leukemia. Leukemia 2:74-78. 12. ISCN (1985): An International System for Human Cytogenetic Nomenclature, Harnden DG, Klinger HP (eds); published in collaboration with Cytogenet Cell Genet (Karger, Basel, 1985); also in Birth Defects: Original Article Series, Vol. 21, No. 1 (March of Dimes Birth Defects Foundation, New York, 1985). 13. Camaschella C, Saglio G. Serra A, Sampietro M, Cappellini MD, Fiorelli G, Cremonesi L, Tedeschi S, Ferrari M (1985): DNA Polymorphism Analysis in the Italian Population and Prenatal Diagnosis of Thalassemia in First Trimester Fetal Diagnosis, M Fraccaro, G Simoni, B Brambati, eds. Springer-Verlag, Berlin. 14. Saglio G, Camaschella C, Giai M, Serra A, Guerrasio A, Peirone B, Gasparini P, Mazza U, Ceppellini R, Biglia N, Cortese P, Sismondi P (1988): Distribution of Ha-ras-1 protooncogene alleles in breast cancer patients and in control population. Breast Cancer Res Treat 11:147-153. 15. Maniatis T, Fritsch EF, Sambrook J (1982): Molecular Cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 16. Harper ME, Saunders GF (1981): Localization of single copy DNA sequences on G-banded h u m a n chromosomes by in sity hybridization. Chromosoma 83:431-439. 17. de Martinville B, Francke U (1983): The H-ras 1 insulin and ~-globin loci map outside the deletion associated with aniridia-Wilms' tumor. Nature 305:641-643. 18. de Martinville B, Giacalone J (1983): Oncogene from h u m a n EJ bladder carcinoma is located on the short arm of chromosome 11. Science 219:498-503. 19. Ohyashiki K, Ohyashiki JH, Sandberg AA, Minowada J, Kinniburg AJ (1987): Loss of a Huets-a allele in h u m a n leukemia cell lines ML-1, -2, and 3 with a chromosome change at 11q24. Cancer Res 47:3842-3846.
