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Trisomy 8 as the sole chromosomal aberration in myelocytic malignancies
Cancer Genetics and Cytogenetics 140 (2003) 66–69
Short communication
Trisomy 8 as the sole chromosomal aberration in myelocytic malignancies: a multicolor and locus-specific fluorescence in situ hybridization study Kajsa Paulssona,*, Thoas Fioretosa, Bodil Strömbecka, Nils Mauritzsonb, Hans J. Tankec, Bertil Johanssona a
Department of Clinical Genetics, University Hospital, SE-221 85 Lund, Sweden b Department of Hematology, University Hospital, SE-221 85 Lund, Sweden c Laboratory for Cytochemistry and Cytometry, Department of Molecular Cell Biology, Leiden University Medical Center, 2333 AL Leiden, The Netherlands Received 6 May 2002; accepted 24 May 2002
Abstract
Trisomy 8 is the most common chromosomal aberration in myelocytic malignancies, occurring both as a sole change as well as in addition to other abnormalities. In spite of this, next to nothing is known about its pathogenetic importance or its molecular genetic consequences. Possible mechanisms involved in the transformation process include dosage effects of genes mapping to chromosome 8 and presence of specific mutations or cryptic fusion genes on the duplicated chromosome. In the latter case, 8 would be secondary to a cryptic primary rearrangement and not involved in leukemogenesis as such, but rather in tumor evolution. Although hidden genetic changes have been found in some trisomies, for example, mutations in KIT in acute myelocytic leukemia (AML) with 4 and in MET in hereditary papillary kidney carcinoma with trisomy 7, none associated with 8 have so far been discovered. To address this issue, we have investigated a total of 13 cases of AML, myelodysplastic syndromes, and chronic myeloproliferative disorders with trisomy 8 as the sole chromosomal anomaly. All cases were studied by combined binary ratio multicolor fluorescence in situ hybridization (FISH) and with FISH using locus-specific probes for both arms of chromosome 8, the subtelomeric regions of 8p and 8q, and the leukemia-associated genes FGFR1, MOZ, ETO, and MYC. No cryptic changes were detected, thus excluding the possibility of gross genetic rearrangements or aberrations involving these loci on chromosome 8. © 2003 Elsevier Science Inc. All rights reserved.
1. Introduction Acquired clonal chromosomal aberrations, balanced as well as unbalanced, are found in 40–50% of acute myelocytic leukemias (AML), myelodysplastic syndromes (MDS), and chronic myeloproliferative disorders (MPD) [1,2]. Whereas balanced rearrangements, mainly translocations and inversions, have been shown to result in the formation of fusion genes with transforming effects [3], the consequences of gains and losses of genetic material, such as monosomies and trisomies, remain enigmatic [4]. Possible underlying mechanisms for the tumor-promoting impact of
* Corresponding author. Tel.: 46-46-17-33-98; fax: 46-46-13-10-61. E-mail address: [email protected] (K. Paulsson).
trisomies include gene dosage effects and presence of specific mutations or cryptic fusion genes on the duplicated chromosomes. In the latter case, trisomies would be secondary changes to a hidden transforming genetic aberration and would not be involved in the transformation process as such, but rather in tumor evolution [5]. Recent findings of cryptic abnormalities in duplicated chromosomes, that is, the mutations in KIT in AML with trisomy 4 [6] and in MET in hereditary papillary kidney carcinoma with trisomy 7 [7] support this hypothesis. For many common trisomies, for example, 8 in malignant hematologic disorders, however, no such hidden anomalies have as yet been identified. Trisomy 8 is the most common aberration in myelocytic malignancies [8], and is present as a sole chromosomal abnormality in 5–7.5% of all cytogenetically abnormal AML, MDS, and MPD [9]. It is also a common secondary change
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K. Paulsson et al. / Cancer Genetics and Cytogenetics 140 (2003) 66–69
in these disorders, suggesting that cryptic primary rearrangements may be present even when 8 is the only cytogenetic finding [5]. This hypothesis is further supported by the facts that the incidence of isolated trisomy 8 shows a marked heterogeneity in AML, MDS, and MPD in relation to gender, age, previous iatrogenic treatment, and morphologic subtype [9], that 8 is a common anomaly in polyclonal hematologic malignancies [10], and that a recent study of the clonal origin of MDS suggests that this change occurs late in the transformation process [11]. To address the possibility that isolated trisomy 8 may be secondary to cryptic rearrangements, we have used FISH, including multicolor-FISH (M-FISH) [12], to search for such hidden changes in AML, MDS, and MPD with 8 as the sole cytogenetically detectable chromosomal aberration. 2. Materials and methods A total of 13 cases of AML, MDS, or MPD with 8 were included in the analyses. Inclusion was based on the following criteria: that trisomy 8 should be the sole chromosomal anomaly, that the abnormal clone was large, and that cells in fixative with good quality metaphases were available. Bone marrow preparations and G-banding had been done according to standard methods, and conventional FISH was performed as described previously [13] on cells that had been stored in fixative (methanol:acetic acid; 3:1) at 20C for 0–9 years. The signals were detected using an Axioplan 2 fluorescence microscope (Carl Zeiss Jena GmbH, Jena, Germany) and analyzed using a CytoVision Ultra system (Applied Imaging, Newcastle, UK).
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For the M-FISH analysis, whole chromosome painting probes for combined binary ratio labeling (COBRA)-FISH were used [12]. In addition, the following probes were applied: partial chromosome painting (PCP) and subtelomeric probes for 8p and 8q (AL Technologies Inc., Arlington, VA, USA; Vysis, Downers Grove, IL, USA), YAC:s 899e2 and 176c9 (CEPH, Paris, France), PAC 164, and LSI c-myc (Vysis). 3. Results and discussion Despite the indications that isolated trisomy 8 may be secondary to cryptic chromosomal rearrangements in myelocytic malignancies [5,9–11], few previous studies have focused on this issue. Langabeer et al. [14] found no evidence for cryptic PML/RARA fusions in AML with 8 as the sole anomaly, and Diaz et al. [15] reported that neither MYC nor MOS, with one exception, were rearranged in various hematologic malignancies. To the best of our knowledge, no previous investigations using M-FISH or locusspecific FISH have been reported to date. In the present study, no hidden anomalies involving chromosome 8 or any other chromosome were found with M-FISH, PC and subtelomeric probes for 8p and 8q, or with locus-specific probes for the leukemia-associated genes FGFR1, MOZ, ETO, and MYC (Table 1), not even among the three polyclonal cases included in the study (cases 3, 4, and 10; Table 1). Spectral karyotyping (SKY) and M-FISH enable labeling and identification of each chromosomal pair, making it possible to detect cryptic rearrangements not visible by conventional banding methods [17]. In the present study, M-FISH did not reveal any additional changes in the 13 cases inves-
Table 1 Clinicial, cytogenetic, and FISH findings in myelocytic malignancies with trisomy 8 as the sole anomaly Patient no. Diagnosis 1 2 3b 4b 5 6 7c 8 9 10 11 12 13
AML-NOS AML-M1 AML-M2 AML-M2 AML-M4 AML-M4 MDS-RA MDS-RA MDS-RARS MDS-RAEB MDS-RAEBt MDS-CMML MDS-CMML MPD-NOS
Sex/ age Previous (y) CT/RT Karyotype F/61 F/65 M/68 M/64 M/85 M/28 M/53 M/75 M/69 F/81 F/82 M/67 M/85 M/74
No No No No No No RT No No CT/RT No RT No
47,XX,8[25] 47,XX,8[22] 47,XY,8[8]/46,XY,del(20)(q11)[4]/46,XY[14] 47,XY,8[16]/46,XY,del(9)(q22)[3]/46,XY[4] 47,XY,8[7]/46,XY[18] 47,XY,8[25] 47,XY,8[10]/46,XY[9] 47,XY,8[15] 47,XY,8[15]/46,XY[10] 4647,XX,8[cp10]/4748,idem,21[cp14] 47,XX,21[5]/48,idem,8[3]/46,XX[4] 47,XY,8[25] 47,XY,8[25] 47,XY,8[23]/46,XY[2]
No. of analyzed metaphases with 8 PCP 8p, subtel 8p, 899e2, 176c9 PAC 164 LSI c-myc COBRA PCP 8q subtel 8q (MOZ, FGFR1) (ETO) (MYC) 4a 4a 2 3 2 4 4 5 5 2
9 3 5 3 6 17 9 23 11 4
5 12 10 11 9 25 10 24 25 13
5 3 4 7 12 6 10 28 10 11
3 3 6 2 4 15 10 16 17 3
10 5 3 6 7 15 5 26 7 11
6 7 4
13 22 11
22 22 23
7 10 11
14 19 12
11 29 23
Metaphases with random losses of signals were excluded. Abbreviations: AML, acute myelocytic leukemia; CMML, chronic myelomonocytic leukemia; COBRA, combined binary ratio labeling (FISH); CT, chemotherapy; F, female; M, male; MDS, myelodysplastic syndromes; MPD, chronic myeloproliferative disorder; NOS, not otherwise specified; RA, refractory anemia; RAEB, refractory anemia with excess of blasts; RAEBt, RAEB in transformation; RARS, refractory anemia with ringed sideroblasts; RT, radiotherapy. a Some of the analyzed metaphases contained random chromosomal losses. b Previously published in Johansson et al. [10]. c Previously published in Mauritzson et al. [16].
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tigated. Although no previous SKY or M-FISH studies have specifically focused on trisomy 8 as the sole anomaly, we have been able to retrieve a total of eight hematologic malignancies, all AML, with isolated 8 that have been included in such investigations [18–20], and in line with our results, no additional aberrations were reported. Because large rearrangements between different chromosomes should have been identified by these techniques, it seems safe to conclude that putative cryptic abnormalities in cases with 8 must involve only one chromosome, or small chromosomal or subtelomeric parts. In this study, PCP probes for 8p and 8q were applied to investigate the first possibility, but without detection of any aberrations, thus excluding the presence of intrachromosomal rearrangements between the two arms of chromosome 8. With regard to minor deletions or duplications, these would not be found using PCP probes or M-FISH, but may be identifiable using genomic microarray with BAC probes [21]. Translocations involving the subtelomeric regions are difficult to detect both with G-banding and SKY/M-FISH, but can be identified by using subtelomeric FISH probes [22,23]. In this investigation, FISH with subtelomeric probes for 8p and 8q did not reveal any changes. Needless to say, abnormalities involving the telomeric regions of other chromosomes may well exist. One way to address this would be to use the newly developed subtelomeric multicolor-FISH (M-TELFISH) methodology [24,25]; in fact, one single case of AML with isolated trisomy 8 was included in the study recently reported by Brown et al. [24], but no additional abnormalities were detected. It is obvious that more cases need to be investigated before any firm conclusions can be drawn. Four genes on chromosome 8 involved in recurrent rearrangements in hematologic malignancies [8] were included in the study, namely FGFR1 and MOZ in 8p11, ETO in 8q22, and MYC in 8q24. Of these, MOZ and ETO were of particular interest because they have previously been reported to be involved in cytogenetically undetectable aberrations [26,27]; however, neither these nor FGFR1 and MYC were rearranged in any of the investigated cases. To the best of our knowledge, no previous studies have focused on cryptic abnormalities in these genes in myelocytic malignancies with 8, except for the previously mentioned Southern blot study of MYC [15]. As regards this gene, it has repeatedly been implied that the tumorigenic effect of trisomy 8 in myelocytic malignancies might be ascribed to its duplication and presumed overexpression [28–30]. If this is correct, then 8 would indeed be a primary cytogenetic event; however, a recent microarray study of AML with isolated 8 revealed a down-regulation of MYC as compared with normal CD34 cells [31]. Taken together, rearrangements or overexpression of MYC does not seem to be of pathogenetic importance in myelocytic malignancies with trisomy 8. The present findings notwithstanding, it cannot be excluded that cryptic rearrangements exist in AML, MDS, and MPD with isolated trisomy 8. Small rearrangements between
chromosomes or even larger anomalies within a chromosome would not be detected by M-FISH, and translocations involving the subtelomeric regions may be overlooked. In addition, locus-specific probes were used only for chromosome 8, but hidden changes could, of course, involve other chromosomes. For example, a cryptic t(7;12)(q36;p13) was recently reported in infant acute lymphoblastic leukemia [32,33], often harboring 8 as an associated secondary change. There may well be other primary rearrangements associated with trisomy 8 waiting to be discovered. Acknowledgments This study was supported by grants from the Swedish Cancer Society. The authors thank Dr. Birg, Marseilles, France for providing the PAC 164 clone. References [1] Mertens F, Johansson B, Heim S, Kristoffersson U, Mitelman F. Karyotypic patterns in chronic myeloproliferative disorders: report on 74 cases and review of the literature. Leukemia 1991;5:214–20. [2] Mauritzson N, Johansson B, Albin M, Billström R, Ahlgren T, Mikoczy Z, Nilsson PG, Hagmar L, Mitelman F. A single-center population-based consecutive series of 1500 cytogenetically investigated adult hematological malignancies: karyotypic features in relation to morphology, age and gender. Eur J Haematol 1999;62:95–102. [3] Rabbitts TH. Chromosomal translocation master genes, mouse models and experimental therapeutics. Oncogene 2001;20:5763–77. [4] Heim S. Is cancer cytogenetics reducible to the molecular genetics of cancer cells? Genes Chromosomes Cancer 1992;5:188–96. [5] Johansson B, Mertens F, Mitelman F. Primary vs. secondary neoplasia-associated chromosomal abnormalities - balanced rearrangements vs. genomic imbalances? Genes Chromosomes Cancer 1996;16:155–63. [6] Beghini A, Ripamonti CB, Castorina P, Pezzetti L, Doneda L, Cairoli R, Morra E, Larizza L. Trisomy 4 leading to duplication of a mutated KIT allele in acute myeloid leukemia with mast cell involvement. Cancer Genet Cytogenet 2000;119:26–31. [7] Zhuang Z, Park W-S, Pack S, Schmidt L, Vortmeyer AO, Pak E, Pham T, Weil RJ, Candidus S, Lubensky IA, Linehan WM, Zbar B, Weirich G. Trisomy 7-harbouring non-random duplication of the mutant MET allele in hereditary papillary renal carcinomas. Nat Genet 1998;20: 66–9. [8] Mitelman F, Johansson B, Mertens F, editors. Mitelman Database of Chromosome Aberrations in Cancer. 2002. http://www.cgap.nci.nih. gov/Chromosomes/Mitelman [9] Paulsson K, Säll T, Fioretos T, Mitelman F, Johansson B. The incidence of trisomy 8 as a sole chromosomal aberration in myeloid malignancies varies in relation to gender, age, prior iatrogenic genotoxic exposure, and morphology. Cancer Genet Cytogenet 2001;130:160–5. [10] Johansson B, Billström R, Broberg K, Fioretos T, Nilsson P-G, Ahlgren T, Malm C, Samuelsson BO, Mitelman F. Cytogenetic polyclonality in hematologic malignancies. Genes Chromosomes Cancer 1999;24:222–9. [11] Nilsson L, Åstrand-Grundström I, Anderson K, Arvidsson I, Hokland P, Bryder D, Kjeldsen L, Johansson B, Hellström-Lindberg E, Hast R, Jacobsen SEW. Involvement and functional impairment of the CD34CD38-Thy-1 hematopoietic stem cell pool in myelodysplatic syndromes with trisomy 8. Blood 2002;100:259–67. [12] Tanke HJ, Wiegant J, van Gijlswijk RPM, Bezrookove V, Pattenier H, Heetebrij RJ, Talman EG, Raap AK, Vrolijk J. New strategy for multi-colour fluorescence in situ hybridisation: COBRA: COmbined Binary RAtio labelling. Eur J Hum Genet 1999;7:2–11.
K. Paulsson et al. / Cancer Genetics and Cytogenetics 140 (2003) 66–69 [13] Gisselsson D. Refined characterisation of chromosome aberrations in tumours by multicolour banding and electronic mapping resources. Methods Cell Sci 2001;23:23–8. [14] Langabeer SE, Grimwade D, Walker H, Rogers JR, Burnett AK, Goldstone AH, Linch DC. A study to determine whether trisomy 8, deleted 9q and trisomy 22 are markers of cryptic rearrangements of PML/RAR, AML1/ETO and CBFB/MYH11, respectively, in acute myeloid leukaemia. Br J Haematol 1998;101:338–40. [15] Diaz MO, Le Beau MM, Harden A, Rowley JD. Trisomy 8 in human hematologic neoplasia and the c-myc and c-mos oncogenes. Leuk Res 1985;9:1437–42. [16] Mauritzson N, Johansson B, Rylander L, Albin M, Strömberg U, Billström R, Ahlgren T, Mikoczy Z, Mitelman F, Hagmar L, Nilsson PG. The prognostic impact of karyotypic subgroups in myelodysplastic syndromes is strongly modified by sex. Br J Haematol 2001;113:347–56. [17] Schröck E, Padilla-Nash H. Spectral karyotyping and multicolor fluorescence in situ hybridization reveal new tumor-specific chromosomal aberrations. Semin Hematol 2000;37:334–47. [18] Mohr B, Bornhäuser M, Thiede C, Schäkel U, Schaich M, Illmer T, Pascheberg U, Ehninger G. Comparison of spectral karyotyping and conventional cytogenetics in 39 patients with acute myeloid leukemia and myelodysplastic syndrome. Leukemia 2000;14:1031–8. [19] Hilgenfeld E, Padilla-Nash H, McNeil N, Knutsen T, Montagna C, Tchinda J, Horst J, Ludwig W-D, Serve H, Büchner T, Berdel WE, Schröck E, Ried T. Spectral karyotyping and fluorescence in situ hybridization detect novel chromosomal aberrations, a recurring involvement of chromosome 21 and amplification of the MYC oncogene in acute myeloid leukaemia M2. Br J Haematol 2001;113:305–17. [20] Kerndrup GB, Kjeldsen E. Acute leukemia cytogenetics: an evaluation of combining G-band karyotyping with multi-color spectral karyotyping. Cancer Genet Cytogenet 2001;124:7–11. [21] Cai W-W, Mao J-H, Chow C-W, Damani S, Balmain A, Bradley A. Genome-wide detection of chromosomal imbalances in tumors using BAC microarrays. Nat Biotechnol 2002;20:393–6. [22] Jaju RJ, Haas OA, Neat M, Harbott J, Saha V, Boultwood J, Brown JM, Pirc-Danoewinata H, Krings BW, Müller U, Morris SW, Wainscoat JS, Kearney L. A new recurrent translocation, t(5;11)(q35; p15.5), associated with del(5q) in childhood acute myeloid leukemia. Blood 1999;94:773–80. [23] Knight SJL, Regan R, Nicod A, Horsley SW, Kearney L, Homfray T, Winter RM, Bolton P, Flint J. Subtle chromosomal rearrangements in children with unexplained mental retardation. Lancet 1999;354:1676–81.
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[24] Brown J, Saracoglu K, Uhrig S, Speicher MR, Eils R, Kearney L. Subtelomeric chromosome rearrangements are detected using an innovative 12-color FISH assay (M-TEL). Nat Med 2001;7:497–501. [25] Brown J, Jawad M, Twigg SRF, Saracoglu K, Sauerbrey A, Thomas AE, Eils R, Harbott J, Kearney L. A cryptic t(5;11)(q35;p15.5) in 2 children with acute myeloid leukemia with apparently normal karyotypes, identified by a multiplex fluorescence in situ hybridization telomere assay. Blood 2002;99:2526–31. [26] Chaffanet M, Mozziconacci M-J, Fernandez F, Sainty D, LafagePochitaloff M, Birnbaum D, Pébusque MJ. A case of inv(8)(p11q24) associated with acute myeloid leukemia involves the MOZ and CBP genes in a masked t(8;16). Genes Chromosomes Cancer 1999;26: 161–5. [27] Harrison CJ, Radford-Weiss I, Ross F, Rack K, le Guyader G, Vekemans M, Macintyre E. Fluorescence in situ hybridization analysis of masked (8;21)(q22;q22) translocations. Cancer Genet Cytogenet 1999; 112:15–20. [28] Blick M, Romero P, Talpaz M, Kurzrock R, Shtalrid M, Andersson B, Trujillo J, Beran M, Gutterman J. Molecular characteristics of chronic myelogenous leukemia in blast crisis. Cancer Genet Cytogenet 1987;27:349–56. [29] Jennings BA, Mills KI. c-myc locus amplification and the acquisition of trisomy 8 in the evolution of chronic myeloid leukaemia. Leuk Res 1998;22:899–903. [30] Oudat R, Khan Z, Glassman AB. Detection of trisomy 8 in Philadelphia chromosome-positive CML patients using conventional cytogenetic and interphase fluorescence in situ hybridization techniques and its relation to c-myc involvement. Ann Clin Lab Sci 2001;31:68–74. [31] Virtaneva K, Wright FA, Tanner SM, Yuan B, Lemon WJ, Caligiuri MA, Bloomfield CD, de la Chapelle A, Krahe R. Expression profiling reveals fundamental biological differences in acute myeloid leukemia with isolated trisomy 8 and normal cytogenetics. Proc Natl Acad Sci USA 2001;98:1124–9. [32] Tosi S, Harbott J, Teigler-Schlegel A, Haas OA, Pirc-Danoewinata H, Harrison CJ, Biondi A, Cazzaniga G, Kempski H, Scherer SW, Kearney L. t(7;12)(q36;p13), a new recurrent translocation involving ETV6 in infant leukemia. Genes Chromosomes Cancer 2000;29:325–32. [33] Slater RM, v Drunen E, Kroes WG, Weghuis DO, van den Berg E, Smit EM, van der Does-van den Berg A, van Wering E, Hählen K, Carroll AJ, Raimondi SC, Beverloo HB. t(7;12)(q36;p13) and t(7; 12)(q32;p13) - translocations involving ETV6 in children 18 months of age or younger with myeloid disorders. Leukemia 2001;15:915–20.
Short communication
Trisomy 8 as the sole chromosomal aberration in myelocytic malignancies: a multicolor and locus-specific fluorescence in situ hybridization study Kajsa Paulssona,*, Thoas Fioretosa, Bodil Strömbecka, Nils Mauritzsonb, Hans J. Tankec, Bertil Johanssona a
Department of Clinical Genetics, University Hospital, SE-221 85 Lund, Sweden b Department of Hematology, University Hospital, SE-221 85 Lund, Sweden c Laboratory for Cytochemistry and Cytometry, Department of Molecular Cell Biology, Leiden University Medical Center, 2333 AL Leiden, The Netherlands Received 6 May 2002; accepted 24 May 2002
Abstract
Trisomy 8 is the most common chromosomal aberration in myelocytic malignancies, occurring both as a sole change as well as in addition to other abnormalities. In spite of this, next to nothing is known about its pathogenetic importance or its molecular genetic consequences. Possible mechanisms involved in the transformation process include dosage effects of genes mapping to chromosome 8 and presence of specific mutations or cryptic fusion genes on the duplicated chromosome. In the latter case, 8 would be secondary to a cryptic primary rearrangement and not involved in leukemogenesis as such, but rather in tumor evolution. Although hidden genetic changes have been found in some trisomies, for example, mutations in KIT in acute myelocytic leukemia (AML) with 4 and in MET in hereditary papillary kidney carcinoma with trisomy 7, none associated with 8 have so far been discovered. To address this issue, we have investigated a total of 13 cases of AML, myelodysplastic syndromes, and chronic myeloproliferative disorders with trisomy 8 as the sole chromosomal anomaly. All cases were studied by combined binary ratio multicolor fluorescence in situ hybridization (FISH) and with FISH using locus-specific probes for both arms of chromosome 8, the subtelomeric regions of 8p and 8q, and the leukemia-associated genes FGFR1, MOZ, ETO, and MYC. No cryptic changes were detected, thus excluding the possibility of gross genetic rearrangements or aberrations involving these loci on chromosome 8. © 2003 Elsevier Science Inc. All rights reserved.
1. Introduction Acquired clonal chromosomal aberrations, balanced as well as unbalanced, are found in 40–50% of acute myelocytic leukemias (AML), myelodysplastic syndromes (MDS), and chronic myeloproliferative disorders (MPD) [1,2]. Whereas balanced rearrangements, mainly translocations and inversions, have been shown to result in the formation of fusion genes with transforming effects [3], the consequences of gains and losses of genetic material, such as monosomies and trisomies, remain enigmatic [4]. Possible underlying mechanisms for the tumor-promoting impact of
* Corresponding author. Tel.: 46-46-17-33-98; fax: 46-46-13-10-61. E-mail address: [email protected] (K. Paulsson).
trisomies include gene dosage effects and presence of specific mutations or cryptic fusion genes on the duplicated chromosomes. In the latter case, trisomies would be secondary changes to a hidden transforming genetic aberration and would not be involved in the transformation process as such, but rather in tumor evolution [5]. Recent findings of cryptic abnormalities in duplicated chromosomes, that is, the mutations in KIT in AML with trisomy 4 [6] and in MET in hereditary papillary kidney carcinoma with trisomy 7 [7] support this hypothesis. For many common trisomies, for example, 8 in malignant hematologic disorders, however, no such hidden anomalies have as yet been identified. Trisomy 8 is the most common aberration in myelocytic malignancies [8], and is present as a sole chromosomal abnormality in 5–7.5% of all cytogenetically abnormal AML, MDS, and MPD [9]. It is also a common secondary change
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K. Paulsson et al. / Cancer Genetics and Cytogenetics 140 (2003) 66–69
in these disorders, suggesting that cryptic primary rearrangements may be present even when 8 is the only cytogenetic finding [5]. This hypothesis is further supported by the facts that the incidence of isolated trisomy 8 shows a marked heterogeneity in AML, MDS, and MPD in relation to gender, age, previous iatrogenic treatment, and morphologic subtype [9], that 8 is a common anomaly in polyclonal hematologic malignancies [10], and that a recent study of the clonal origin of MDS suggests that this change occurs late in the transformation process [11]. To address the possibility that isolated trisomy 8 may be secondary to cryptic rearrangements, we have used FISH, including multicolor-FISH (M-FISH) [12], to search for such hidden changes in AML, MDS, and MPD with 8 as the sole cytogenetically detectable chromosomal aberration. 2. Materials and methods A total of 13 cases of AML, MDS, or MPD with 8 were included in the analyses. Inclusion was based on the following criteria: that trisomy 8 should be the sole chromosomal anomaly, that the abnormal clone was large, and that cells in fixative with good quality metaphases were available. Bone marrow preparations and G-banding had been done according to standard methods, and conventional FISH was performed as described previously [13] on cells that had been stored in fixative (methanol:acetic acid; 3:1) at 20C for 0–9 years. The signals were detected using an Axioplan 2 fluorescence microscope (Carl Zeiss Jena GmbH, Jena, Germany) and analyzed using a CytoVision Ultra system (Applied Imaging, Newcastle, UK).
67
For the M-FISH analysis, whole chromosome painting probes for combined binary ratio labeling (COBRA)-FISH were used [12]. In addition, the following probes were applied: partial chromosome painting (PCP) and subtelomeric probes for 8p and 8q (AL Technologies Inc., Arlington, VA, USA; Vysis, Downers Grove, IL, USA), YAC:s 899e2 and 176c9 (CEPH, Paris, France), PAC 164, and LSI c-myc (Vysis). 3. Results and discussion Despite the indications that isolated trisomy 8 may be secondary to cryptic chromosomal rearrangements in myelocytic malignancies [5,9–11], few previous studies have focused on this issue. Langabeer et al. [14] found no evidence for cryptic PML/RARA fusions in AML with 8 as the sole anomaly, and Diaz et al. [15] reported that neither MYC nor MOS, with one exception, were rearranged in various hematologic malignancies. To the best of our knowledge, no previous investigations using M-FISH or locusspecific FISH have been reported to date. In the present study, no hidden anomalies involving chromosome 8 or any other chromosome were found with M-FISH, PC and subtelomeric probes for 8p and 8q, or with locus-specific probes for the leukemia-associated genes FGFR1, MOZ, ETO, and MYC (Table 1), not even among the three polyclonal cases included in the study (cases 3, 4, and 10; Table 1). Spectral karyotyping (SKY) and M-FISH enable labeling and identification of each chromosomal pair, making it possible to detect cryptic rearrangements not visible by conventional banding methods [17]. In the present study, M-FISH did not reveal any additional changes in the 13 cases inves-
Table 1 Clinicial, cytogenetic, and FISH findings in myelocytic malignancies with trisomy 8 as the sole anomaly Patient no. Diagnosis 1 2 3b 4b 5 6 7c 8 9 10 11 12 13
AML-NOS AML-M1 AML-M2 AML-M2 AML-M4 AML-M4 MDS-RA MDS-RA MDS-RARS MDS-RAEB MDS-RAEBt MDS-CMML MDS-CMML MPD-NOS
Sex/ age Previous (y) CT/RT Karyotype F/61 F/65 M/68 M/64 M/85 M/28 M/53 M/75 M/69 F/81 F/82 M/67 M/85 M/74
No No No No No No RT No No CT/RT No RT No
47,XX,8[25] 47,XX,8[22] 47,XY,8[8]/46,XY,del(20)(q11)[4]/46,XY[14] 47,XY,8[16]/46,XY,del(9)(q22)[3]/46,XY[4] 47,XY,8[7]/46,XY[18] 47,XY,8[25] 47,XY,8[10]/46,XY[9] 47,XY,8[15] 47,XY,8[15]/46,XY[10] 4647,XX,8[cp10]/4748,idem,21[cp14] 47,XX,21[5]/48,idem,8[3]/46,XX[4] 47,XY,8[25] 47,XY,8[25] 47,XY,8[23]/46,XY[2]
No. of analyzed metaphases with 8 PCP 8p, subtel 8p, 899e2, 176c9 PAC 164 LSI c-myc COBRA PCP 8q subtel 8q (MOZ, FGFR1) (ETO) (MYC) 4a 4a 2 3 2 4 4 5 5 2
9 3 5 3 6 17 9 23 11 4
5 12 10 11 9 25 10 24 25 13
5 3 4 7 12 6 10 28 10 11
3 3 6 2 4 15 10 16 17 3
10 5 3 6 7 15 5 26 7 11
6 7 4
13 22 11
22 22 23
7 10 11
14 19 12
11 29 23
Metaphases with random losses of signals were excluded. Abbreviations: AML, acute myelocytic leukemia; CMML, chronic myelomonocytic leukemia; COBRA, combined binary ratio labeling (FISH); CT, chemotherapy; F, female; M, male; MDS, myelodysplastic syndromes; MPD, chronic myeloproliferative disorder; NOS, not otherwise specified; RA, refractory anemia; RAEB, refractory anemia with excess of blasts; RAEBt, RAEB in transformation; RARS, refractory anemia with ringed sideroblasts; RT, radiotherapy. a Some of the analyzed metaphases contained random chromosomal losses. b Previously published in Johansson et al. [10]. c Previously published in Mauritzson et al. [16].
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tigated. Although no previous SKY or M-FISH studies have specifically focused on trisomy 8 as the sole anomaly, we have been able to retrieve a total of eight hematologic malignancies, all AML, with isolated 8 that have been included in such investigations [18–20], and in line with our results, no additional aberrations were reported. Because large rearrangements between different chromosomes should have been identified by these techniques, it seems safe to conclude that putative cryptic abnormalities in cases with 8 must involve only one chromosome, or small chromosomal or subtelomeric parts. In this study, PCP probes for 8p and 8q were applied to investigate the first possibility, but without detection of any aberrations, thus excluding the presence of intrachromosomal rearrangements between the two arms of chromosome 8. With regard to minor deletions or duplications, these would not be found using PCP probes or M-FISH, but may be identifiable using genomic microarray with BAC probes [21]. Translocations involving the subtelomeric regions are difficult to detect both with G-banding and SKY/M-FISH, but can be identified by using subtelomeric FISH probes [22,23]. In this investigation, FISH with subtelomeric probes for 8p and 8q did not reveal any changes. Needless to say, abnormalities involving the telomeric regions of other chromosomes may well exist. One way to address this would be to use the newly developed subtelomeric multicolor-FISH (M-TELFISH) methodology [24,25]; in fact, one single case of AML with isolated trisomy 8 was included in the study recently reported by Brown et al. [24], but no additional abnormalities were detected. It is obvious that more cases need to be investigated before any firm conclusions can be drawn. Four genes on chromosome 8 involved in recurrent rearrangements in hematologic malignancies [8] were included in the study, namely FGFR1 and MOZ in 8p11, ETO in 8q22, and MYC in 8q24. Of these, MOZ and ETO were of particular interest because they have previously been reported to be involved in cytogenetically undetectable aberrations [26,27]; however, neither these nor FGFR1 and MYC were rearranged in any of the investigated cases. To the best of our knowledge, no previous studies have focused on cryptic abnormalities in these genes in myelocytic malignancies with 8, except for the previously mentioned Southern blot study of MYC [15]. As regards this gene, it has repeatedly been implied that the tumorigenic effect of trisomy 8 in myelocytic malignancies might be ascribed to its duplication and presumed overexpression [28–30]. If this is correct, then 8 would indeed be a primary cytogenetic event; however, a recent microarray study of AML with isolated 8 revealed a down-regulation of MYC as compared with normal CD34 cells [31]. Taken together, rearrangements or overexpression of MYC does not seem to be of pathogenetic importance in myelocytic malignancies with trisomy 8. The present findings notwithstanding, it cannot be excluded that cryptic rearrangements exist in AML, MDS, and MPD with isolated trisomy 8. Small rearrangements between
chromosomes or even larger anomalies within a chromosome would not be detected by M-FISH, and translocations involving the subtelomeric regions may be overlooked. In addition, locus-specific probes were used only for chromosome 8, but hidden changes could, of course, involve other chromosomes. For example, a cryptic t(7;12)(q36;p13) was recently reported in infant acute lymphoblastic leukemia [32,33], often harboring 8 as an associated secondary change. There may well be other primary rearrangements associated with trisomy 8 waiting to be discovered. Acknowledgments This study was supported by grants from the Swedish Cancer Society. The authors thank Dr. Birg, Marseilles, France for providing the PAC 164 clone. References [1] Mertens F, Johansson B, Heim S, Kristoffersson U, Mitelman F. Karyotypic patterns in chronic myeloproliferative disorders: report on 74 cases and review of the literature. Leukemia 1991;5:214–20. [2] Mauritzson N, Johansson B, Albin M, Billström R, Ahlgren T, Mikoczy Z, Nilsson PG, Hagmar L, Mitelman F. A single-center population-based consecutive series of 1500 cytogenetically investigated adult hematological malignancies: karyotypic features in relation to morphology, age and gender. Eur J Haematol 1999;62:95–102. [3] Rabbitts TH. Chromosomal translocation master genes, mouse models and experimental therapeutics. Oncogene 2001;20:5763–77. [4] Heim S. Is cancer cytogenetics reducible to the molecular genetics of cancer cells? Genes Chromosomes Cancer 1992;5:188–96. [5] Johansson B, Mertens F, Mitelman F. Primary vs. secondary neoplasia-associated chromosomal abnormalities - balanced rearrangements vs. genomic imbalances? Genes Chromosomes Cancer 1996;16:155–63. [6] Beghini A, Ripamonti CB, Castorina P, Pezzetti L, Doneda L, Cairoli R, Morra E, Larizza L. Trisomy 4 leading to duplication of a mutated KIT allele in acute myeloid leukemia with mast cell involvement. Cancer Genet Cytogenet 2000;119:26–31. [7] Zhuang Z, Park W-S, Pack S, Schmidt L, Vortmeyer AO, Pak E, Pham T, Weil RJ, Candidus S, Lubensky IA, Linehan WM, Zbar B, Weirich G. Trisomy 7-harbouring non-random duplication of the mutant MET allele in hereditary papillary renal carcinomas. Nat Genet 1998;20: 66–9. [8] Mitelman F, Johansson B, Mertens F, editors. Mitelman Database of Chromosome Aberrations in Cancer. 2002. http://www.cgap.nci.nih. gov/Chromosomes/Mitelman [9] Paulsson K, Säll T, Fioretos T, Mitelman F, Johansson B. The incidence of trisomy 8 as a sole chromosomal aberration in myeloid malignancies varies in relation to gender, age, prior iatrogenic genotoxic exposure, and morphology. Cancer Genet Cytogenet 2001;130:160–5. [10] Johansson B, Billström R, Broberg K, Fioretos T, Nilsson P-G, Ahlgren T, Malm C, Samuelsson BO, Mitelman F. Cytogenetic polyclonality in hematologic malignancies. Genes Chromosomes Cancer 1999;24:222–9. [11] Nilsson L, Åstrand-Grundström I, Anderson K, Arvidsson I, Hokland P, Bryder D, Kjeldsen L, Johansson B, Hellström-Lindberg E, Hast R, Jacobsen SEW. Involvement and functional impairment of the CD34CD38-Thy-1 hematopoietic stem cell pool in myelodysplatic syndromes with trisomy 8. Blood 2002;100:259–67. [12] Tanke HJ, Wiegant J, van Gijlswijk RPM, Bezrookove V, Pattenier H, Heetebrij RJ, Talman EG, Raap AK, Vrolijk J. New strategy for multi-colour fluorescence in situ hybridisation: COBRA: COmbined Binary RAtio labelling. Eur J Hum Genet 1999;7:2–11.
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