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The influence of different chromosomal aberrations on molecular cytogenetic parameters in chronic lymphocytic leukemia
Cancer Genetics and Cytogenetics 167 (2006) 145–149
The influence of different chromosomal aberrations on molecular cytogenetic parameters in chronic lymphocytic leukemia A. Amiela,b,*, L. Leopoldc, N. Gronichd, M. Yuklad, M.D. Fejgina,c, M. Lishnerc,d a
Genetic Institute, Meir Hospital, Kfar-Saba 44281, Israel b Bar Ilan University, Ramat-Gan, Israel c Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel d Department of Medicine and Hematology, Sapir Medical Center, Kfar-Saba, Israel Received 21 October 2005; received in revised form 28 November 2005; accepted 29 November 2005
Abstract
B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia of adults in Western countries. The most frequent recurring chromosomal aberrations identified in B-CLL patients are trisomy 12 and deletions of 13q, 17p, and 11q. Cases with deletions of 11q and 17p have a poor prognosis, whereas cases with deletions in 13q have a favorable prognosis. It was previously shown that CLL patients with trisomy 12 and del(13)(q14) have a higher rate of asynchronous replication of normal structural genes when compared to those with normal karyotypes. We studied the replication pattern of the structural locus 21q22 and the imprinted gene SNRPN and its telomere (15qter) and the random aneuploidy of chromosomes 9 and 18 in CLL patients with trisomy 12 and deletions of 11q and 17p, and compared the results to those of CLL patients without these aberrations and to healthy controls. Random aneuploidy rate was higher in the group of patients with trisomy 12 as compared to all other groups. The replication pattern with higher asynchronous pattern was found in both aberration groups compared to the CLL patients without the aberrations and to the control group with involvement of 21q22 and 15qter, whereas the highest synchronous group was found in the 2 aberrations CLL patient groups compared to the other groups with the imprinted locus SNRPN. The existence and significance of chromosomal aberrations in CLL have a deleterious effect on the processes of cell cycle and gene replication and may have biological and prognostic implications. Ó 2006 Elsevier Inc. All rights reserved.
1. Introduction B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia of adult populations in Western countries [1]. The morphology reveals a monomorphic small round cell population of B-lymphocytes. The disease tends to affect older patients, with a median age of 65 years usually cited [2]. The gender ratio varies from approximately 1.5 to 2.1 males to females [2]. CLL carries a variable prognosis, which is associated with various parameters including genetic aberrations. Classical cytogenetic analyses of bone marrow cells of BCLL patients are often hampered by low mitotic activity of malignant B-cells in culture, and normal chromosome results are obtained in the most patients. Molecular cytogenetic methods, especially fluorescence in situ hybridization
* Corresponding author: Tel.: 1972-9-737-2220; fax: 1972-9-7471296. E-mail address: [email protected] (A. Amiel). 0165-4608/06/$ – see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2005.11.019
(FISH), have enlarged the spectrum of genomic aberrations detected in B-CLL [3]. The most frequent recurring chromosomal aberrations identified in B-CLL patients are trisomy 12 and deletions of 13q, 17p, and 11q [3,4]. Patients with deletion 13q usually have a favorable prognosis and longer survival time, but patients with del(17p) display treatment failure in comparison to patients with del(11)(q22~q23) and trisomy 12 [3]. Deletion (11)(q22~q23) is characteristic of patients with poor prognosis [1,3,4]. The aneuploidy-cancer theory proposes that cancer is caused by the abnormal dosage of thousands of normal genes. This abnormal dosage of genes is generated by the gain or loss of specific chromosomes or segments of chromosomes (alias aneuploidy) [5]. However, the state of aneuploidy destabilizes chromosomes and genes because it unbalances highly conserved teams of proteins that segregate, synthesize, and repair chromosomes. Thus, the inherent instability of aneuploidy catalyzes a chain reaction of chromosome reassortments and rearrangements [5–9]. The theory predicts that chromosomal and genetic
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A. Amiel et al. / Cancer Genetics and Cytogenetics 167 (2006) 145–149
instability is proportional to the degree of aneuploidy and to the types of chromosomes that are unbalanced; this was also confirmed by Fabarius et al. [10]. Numerous studies have demonstrated close association between the replication timing of a given chromosome region during S-phase and transcriptional activity of genes in this region. Hence, expressed loci replicate early in Sphase while silent loci replicate late [11–14]. By using this technique in human somatic cells, it has been convincingly demonstrated that alleles of genes which exhibit allelespecific expression (mono-allelic mode of expression), such as imprinted genes [15], genes subjected to X-chromosome inactivation [16] and olfactory receptor genes [17] replicate asynchronously, whereas alleles which are expressed concomitantly (biallelic mode of expression) replicate highly synchronously [14,18–20]. There is also evidence that alleles of loci, which normally replicate synchronously such as those associated with cell proliferation (TP53, HER2/ neu, and C-MYC ) or chromosome segregation, replicate asynchronously when present in cancer cells [19,21–23]. Some suggest that these alleles behave like the heterochromatic region and replicate in the late S-phase, whereas other believe that they replicate in the early S-phase or randomly during all the S-phase [24–26]. The aim of this study was to analyze whether CLL patients with different cytogenetic aberrations also differ in the parameter of spontaneous aneuploidy. In addition, we assessed the replication status of the CLL patients with and without the examined aberrations, with imprinted locus SNRPN and telomere 15qter, and the 21q22 locus.
2. Materials and methods The study was approved by the local ethical committee. Leukocytes from 54 CLL patients aged 49–88 years at both diagnosis and during treatment and followed at the Hematology Unit in Meir Medical Center were studied and compared to 7 healthy controls of matched age. The diagnosis of CLL was based on lymphocytosis greater than 5x106/mL and immunophenotyping that confirmed the monoclonal origin of the B-lymphocytes. Peripheral blood samples were obtained and incubated for short-term culture in an F10-supplemented medium in a 37 C moist chamber for 72 hours. The supplemented medium contained 20% FCS, lipopolysaccharide (40 mg/mL, 10 units/mL heparin, and 1% antibiotics. The leukocytes from the healthy controls were incubated in the F-10 supplemented medium contained PHA instead of LPS for 72 hours. After incubation, colchicine (final concentration 0.1 mg/mL) was added to the cultures for 1 hour, followed by hypotonic treatment (0.075 M KCl and 37 C for 15 minutes) and 4 washes, each with a fresh cold 3:1 methanolacetic acid solution. The lymphocyte suspensions of the three samples were stored at –4 C.
2.1. Probes For the random aneuploidy the Abbott Vysis probe, CEP 18 spectrum green (Cat No. 5J10-18) and CEPg spectrum orange (Cat No. 6J36-09). For the aberration detection, the following Abbot Vysis probes were used: CEP 12 spectrum (Cat No. 7J20-12); LSI ATM (ataxia telangiectasia mutation at 11q22.3) SpectrumOrange (Cat No. 5J6401); and LSI p53 (17p13.1) SpectrumOrange (Cat No. 5J52-01). For the replication pattern, the following probes were used: SNRPN (Small Nuclear Ribonucleoprotein Polypeptide N) imprinting center - Red Fluorophore with 15qter control probe green fluorophore (Cat No. LPU005, cytocell); and the Abbott Vysis probe 21q22 (Cat No. 8-5JB13-02). 2.2. Specimen pretreatment prior to co-denaturation The pretreatment procedure is highly recommended by Abbott/Vysis protocol prior to hybridization of ToTelVysion probes on lymphocyte specimens when using co-denaturation. The purpose of this procedure is to make the chromosomal DNA accessible for hybridization and to protect the morphology of the chromosomes from the co-denaturation process. 2.3. FISH technique Fresh slide preads were denatured for 2 minutes in 70% formamide standard saline citrate (2SSC) at 70 C and dehydrated in a graded ethanol series. The probe mix was then applied to air-warmed slides (30 mL, mix sealed under a 24x50 mm glass cover slip) and hybridized from 18 hours at 37 C in moist chamber. Following hybridization, the slides were washed in 50% formamide 2XSSC for 20 minutes at 43 C, rinsed in two changes of 2XSSC at 37 C for 4 minutes each, and placed in 0.05% Tween 20 (Sigma, Rehovot, Israel). For FISH analysis, the slides were counterstained in 4,6 diamidino-z-phenyindole (Sigma) antifade solution and analyzed for simultaneous viewing of FITC (Fluorescein Isothiocyanate), Texas Red, and 4,6 diamidinoz-phenyindole with an imaging processing system (Applied Imaging, Santa Clara, Ca.). 2.4. Cytogenetic evaluation Probes for chromosomes 9 and 18 were used for the detection of aneuploidy. For each cell, we recorded the number of hybridization signals. The rate of aneuploidy was inferred from the fraction of cells revealing 1, 3, or more hybridization signals per cell. For the replication pattern interphase cells with 2 hybridization signals were analyzed for each probe. The cells were classified into three categories according to Selig et al. [14]. The samples were scored ‘‘blindly’’. The level of synchrony in replication timing was derived from the fraction of SD cells.
A. Amiel et al. / Cancer Genetics and Cytogenetics 167 (2006) 145–149
2.5. Statistical analysis Two 2-sample t-tests were applied for testing differences between the study groups for quantitative parameters. All tests were two-tailed and a p-value of 0.05 or less was considered statistically significant. We used Microsoft Excel software. 3. Results For the detection of the various aberrations, FISH with CEP12, LS1p53 (17p13.1), and the ATM locus (11q22.3) was performed. Ten (18.5%) out of the 54 CLL patients had trisomy 12, seven (13%) had deletion at TP53, and three (5.5%) had a deletion at the ATM locus. Two patients had both trisomy 12 and 17p deletion. One patient carried 2 deletions: at 17p and 11q. Because of the low number of individuals with the 11q and 17p deletions, we combined them into one group. We found higher aneuploidy rate (monosomy, trisomy, and tetrasomy) in the CLL study group compared to control group ( p ! 0.01; Tables 1 and 2). In the CLL group with normal FISH findings, trisomy and tetrasomy were significantly lower than the trisomy 12 study group with both chromosome analyzed ( p ! 0.05; Tables 1 and 2). Also the rate of monosomy, trisomy, and Table 1 Frequency of aneuploidy with chromosomes 9 and 18 in the control group(1), CLL patient without diagnosed chromosomal aberrations(2), CLL patients with trisomy 12(3) and CLL patients with a deletions of 17p and/or 11q(4) Average 6 standard deviation (%)
Number of samples Chromosome 9 1 8
2
10
3
9
4
8
Chromosome 18 1 8
2
10
3
9
4
8
Monosomy Disomy Trisomy and Monosomy Disomy Trisomy and Monosomy Disomy Trisomy and Monosomy Disomy Trisomy and Monosomy Disomy Trisomy and Monosomy Disomy Trisomy and Monosomy Disomy Trisomy and Monosomy Disomy Trisomy and
Tetrasomy
Tetrasomy
Tetrasomy
Tetrasomy
Tetrasomy
Tetrasomy
Tetrasomy
Tetrasomy
2.62 6 1.19 97.33 6 1.16 0.17 6 0.36 9.89 6 2.85 87.21 6 3.36 2.84 6 1.66 15.54 6 3.58 79.08 6 2.72 4.90 6 2.33 18.27 6 3.88 77.41 6 3.98 4.28 6 1.88 3 6 1.48 97.20 6 1.53 0.17 6 0.31 8.87 6 2.54 87.86 6 2.99 3.21 6 2.09 14.33 6 4.13 79.28 6 3.29 6.35 6 2.36 19.87 6 3.40 76.34 6 2.66 3.75 6 1.59
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Table 2 Statistical analyses of the average of aneuploidy in chromosomes 9 and 18 between all the different groups: (1) Control group, (2) CLL patients without of the chromosomal aberration, (3) CLL patients with trisomy 12, (4) CLL patient with a deletions of 17p and/or 11q. The numbers in the tables represent the p value of two tail-t-test Comparison between the experimental groups Chromosome 9 group (2) compared group (3) compared group (4) compared group (2) compared group (2) compared group (3) compared Chromosome 18 group (2) compared group (3) compared group (4) compared group (2) compared group (2) compared group (3) compared
Trisomy and Monosomy Disomy Tetrasomy
to to to to to to
group group group group group group
(1) (1) (1) (3) (4) (4)
0 0 0 0.0018 0.0002 0.1546
0 0 0 0 0.0001 0.3406
0.0006 0.0003 0.0004 0.0449 0.112 0.5511
to to to to to to
group group group group group group
(1) (1) (1) (3) (4) (4)
0 0 0 0.0045 0 0.0085
0 0 0 0 0 0.0598
0.0012 0 0 0.0075 0.541 0.0176
tetrasomy with chromosome 18 was significantly lower in the group with both deletions (11q and 17p) compared to the group with trisomy 12 ( p ! 0.01; Tables 1 and 2). The synchronous replication patterns of the 21q22 and 15qter were changed to asynchronous pattern (SD) on behalf of the SS pattern. In all CLL patients compared to control group the SD pattern was significantly higher. In the patients with abnormal FISH the rate of asynchrony was significantly higher then in those with normal FISH (Fig. 1a-c, Table 2). When the imprinted locus SNRPN was applied, a trend from asynchronous to synchronous replication in the CLL patients compared to controls was found, whereas in both aberrations groups the synchronous pattern was higher than the CLL group without aberrations ( p ! 0.01; Fig. 1b, Table 2). In all CLL patients, SS pattern was increased on behalf of the SD group (with the SNRPN locus), which means that one allele replicated later. We also found that the 15qter subtelomeric region was modifying the high synchronization pattern in the normal control group, to a synchronization pattern in all CLL patient group, and was the same in the groups with and without the aberrations (Table 2). The asynchrony rate (SD) was increased on behalf of the DD pattern, which means that one allele is replicating later on (Fig. 1c).
4. Discussion The random aneuploidy rate was significantly higher in patients with trisomy 12 compared to the patients with the deletions 11q and 17p and to CLL patients without aberrations. In addition, patients with a 17p deletion have the poorest outcome, followed by 11q deletion and trisomy 12 (that goes with the survival rate), it is possible that trisomy 12, which is a more serious aberration than a local deletion
A. Amiel et al. / Cancer Genetics and Cytogenetics 167 (2006) 145–149
148
a
Locus 21q22.
SD DD SS
CLL with a deletion of 17p or 11q
b
CLL with trisomy 12
CLL without chromosomal aberration
cell frequency (%)
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% control group
Locus SNRPN 100% 90% 70% 60%
SD DD SS
50% 40% 30% 20%
cell frequency (%)
80%
10% 0% CLL with a deletion of 17p or 11q
c
CLL with trisomy 12
CLL without aberration
control group
Locus 15qter.
SD DD SS
CLL with a deletion of 17p or 11q
CLL with trisomy 12
CLL without aberration
cell frequency (%)
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% control group
Fig. 1. Replication pattern of CLL patients and control group with the different loci analyzed.
has more effect on the nearest environment (cells) on the cell cycle progression and thus on chromosome segregation than on the disease phenotype. On the other hand nonexpression of specific loci (TP53 and ATM ) have more serious effect on the disease phenotype and progression [1,3,4]. In a study of gene expression a majority of the significant changes in gene expression were associated with the 11q23 deletion. Hierarchical clustering of statistically significant genes confirmed the similarity in gene expression among cases with the 11q23 deletion and pooled B-cell malignant cell lines. All the other CLL samples with 17p13 deletion, trisomy 12, 13q14 deletion, or normal karyotype were distributed randomly according to gene expression. Therefore, it may be concluded that certain chromosome abnormalities, such as the 11q23 deletion, may not only be markers of poor prognosis, but through their genetic lesion, drive tumor progression by a distinct transcriptional pathway [4]. The asynchronization rate was significantly different between the CLL patients without the examined aberrations
and both groups of aberrations with no difference between the two aberration groups, as was found before [27]. The existence of any of these aberrations has an unfavorable effect on the process of normal gene replication [27]. According to our results, it seems that the mechanisms of cell cycle progression and gene replication control are segregated with different sensitivity to the specific aberration. We found that the 15qter locus is a normal structural behaving locus, which replicates normally in a bi-allelic manner. In CLL patients, the mode of replication resembles the asynchronous pattern groups, similar to the normal structural locus 21q22. It seems that more than one mechanism is involved in the replicating control, which brings about the change from synchronous to asynchronous pattern in cancer cells. While in the 21q22 locus one of the alleles replicated earlier than anticipated in these patients, in the 15qter locus one allele replicated later than anticipated in the same group of patients. The imprinted gene SNPRN, which in normal control cells replicates in non-Mendelian manner (asynchronous) [28,29], changed the pattern of replication
A. Amiel et al. / Cancer Genetics and Cytogenetics 167 (2006) 145–149
towards synchronous manner in CLL patients, a change that had been previously described in cancer patients [28,29]. In both SNRPN and the sub-telomeric group 15qter (both on chromosome 15) one allele replicated later (the same mechanism). It is not clear whether this happened randomly or whether in this condition the subtelomeric loci mimic the behavior of the other loci on the same chromosome. Additional studies of the replication pattern of the different subtelomeric region are needed in order to find whether they share a common mechanism of replication mode in normal and cancer cells. In summary, the confirmed chromosomal aberrations in CLL disease have a deleterious and differential effect on cell cycle and gene replication processes and may have biological and prognostic implications. References [1] Goorha S, Glenn MJ, Drozd-Borysiuk E, Chen Z. A set of commercially available fluorescent in-situ hybridization probes efficient detects cytogenetic abnormalities in patients with chronic lymphocytic leukemia. Genet Med 2004;6:48–53. [2] Glassman AB, Hayes KJ. The value of fluorescence in situ hybridization in the diagnosis and prognosis of chronic lymphocytic leukemia. Cancer Genet Cytogenet 2005;158:88–91. [3] Sindelarova L, Michalova K, Zemanova Z, Ransdorfova S, Brezinova J, Pekova S, Schwarz J, Karban J, Cmunt E. Incidence of chromosomal anomalies detected with FISH and their clinical correlations in B-chronic lymphocytic leukemia. Cancer Genet Cytogenet 2005;160:27–34. [4] Dickinson JD, Smith LM, Sanger WG, Zhou G, Townley P, Lynch JC, Pavletic S, Bierman PJ, Joshi SS. Unique gene expression and clinical characteristics are associated with the 11q23 deletion in chronic lymphocytic leukemia. Brit J Haematol 2005;128:460–71. [5] Duesberg P, Rasnick D. Aneuploidy, the somatic mutation that makes cancer a species of its own. Cell Motil Cytoskel 2000;47:81–107. [6] Li R, Yerganian G, Duesberg P, Kraemer A, Willer A, Rausch C, Hechtmann R. Aneuploidy correlated 100% with chemical transformation of Chinese hamster cells. Proc Natl Acad Sci USA 1997; 94:14506–11. [7] Brinkley BR, Goepfert TM. Supernumerary centrosomes and cancer: Boveri’s hypothesis resurrected. Cell Motil Cytoskeleton 1998;41: 281–8. [8] Duesberg P, Rausch C, Rasnick D, Hehlmann R. Genetic instability of cancer cells is proportional to their degree of aneuploidy. Proc Natl Acad Sci USA 1998;95:13692–7. [9] Rasnick D, Duesberg P. How aneuploidy affects metabolic control and causes cancer. Biochem J 1993;340:621–30. [10] Fabarius A, Willer A, Yerganian G, Hehlmann R, Duesberg P. Specific aneusomies in Chinese hamster cell at different stages of neoplastic transformation, initiated by nitrosomethylurea. Proc Natl Acad Sci USA 2002;99:6773–83. [11] Goldman MA, Holmquist GP, Gray MC, Caston LA, Nag A. Replication timing of mammalian genes and middle repetitive sequences. Science 1984;224:686–92.
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[12] Holmquist GP. Role of replication time in the control of tissue specific gene expression. Am J Hum Genet 1987;40:151–73. [13] Hatton KS, Dhar VH, Brwon EH. Replication program of active and inactive multigene families in mammalian cells. Mol Cell Biol 1988;8:2149–58. [14] Selig S, Okumura K, Ward DC, Cedar H. Delineation of DNA replication time zones by fluorescence in situ hybridization. EMBO J 1992;11:1217–25. [15] Kitsberg D, Selig S, Brandeir M, Simon I, Keshet I, Driscoll DJ, Nichols RD, Cedar H. Allele-specific replication timing of imprinted gene regions. Nature 1993;364:459–63. [16] Boggs BA, Chinault AC. Analysis of replication timing properties of human X-chromosomal loci by fluorescence in situ hybridization. Proc Natl Acad Sci USA 1994;91:6083–7. [17] Chess A, Simon I, Cedar H, Axel R. Allele inactivation regulates olfactory receptor gene expression. Cell 1994;78:823–34. [18] Amiel A, Kirgner I, Gaber E, Manor Y, Fejgin M, Lishner M. Replication pattern in cancer. Asynchronous replication in multiple myeloma and in monoclonal gammopathy. Cancer Genet Cytogenet 1999;108:32–7. [19] Amiel A, Kolodizner T, Fishman A, Gaber E, Klein Z, Beyth Y, Fejgin MD. Replication pattern of the p53 and 21q22 loci in the premalignant and malignant stages of carcinoma of the cervix. Cancer 1998;83:1966–71. [20] Amiel A, Kitai Y, Fejgin M, Lishner M. Replication status as a marker for predisposition for lymphoma in patients with chronic hepatitis C with and without cryoglobulinemia. Exp Hematol 2000;2:156–60. [21] Amiel A, Litmanovich T, Gaber E, Lishner M, Avivi L, Fejgin MD. Asynchronous replication of p53 and 21q22 loci in chronic lymphocytic leukemia. Hum Genet 1997;101:219–22. [22] Amiel A, Litmanovitch T, Lishner M, Mor A, Gaber E, Tangi I, Fejgin M, Avivi L. Temporal differences in replication timing of homologous loci in malignant cells derived from CML and lymphoma patients. Genes Chromosomes Cancer 1998;22:225–31. [23] Amiel A, Avivi L, Gaber E, Fejgin MD. Asynchronous replication of allelic loci in Down syndrome. Eur J Hum Genet 1998;6:359–64. [24] Wright WE, Tesmer VM, Liao ML, Shay JW. Normal human telomeres are not late replicating. Exp Cell Res 1999;251:492–9. [25] Hultdin M, Gronlund KF, Norrback T, Just KT, Roos G. Replication timing of human telomeric DNA and other repetitive sequences analyzed by fluorescence in situ hybridization and flow cytometry. Exp Cell Res 2001;271:223–9. [26] Ofir R, Yalon-Hacohen M, Segev Y, Schultz A, Skorecki KL, Selig S. Replication and/or separation of some human telomeres is delayed beyond S-phase in pre-senescent cells. Chromosoma 2002;111: 147–5. [27] Amiel A, Elis A, Sherker S, Gaber E, Manor Y, Fejgin MD. The influence of cytogenetic aberrations on gene replication in CLL patients. Cancer Genet Cytogenet 2001;125:81–6. [28] Dotan ZA, Dotan A, Litmanovitch T, Ravia Y, Oniashvili N, Leibovitch I, Ramon J, Avivi L. Modification in the inherent mode of allelic replication in lymphocytes of patients suffering from renal cell carcinoma: a novel genetic alteration associated with malignancy. Genes Chromosomes Cancer 2000;27:270–7. [29] Dotan ZA, Dotan A, Ramon J, Avivi L. Altered mode of allelic replication accompanied by aneuploidy in peripheral blood lymphocytes of prostate cancer patients. Int J Cancer 2004;111:60–6.
The influence of different chromosomal aberrations on molecular cytogenetic parameters in chronic lymphocytic leukemia A. Amiela,b,*, L. Leopoldc, N. Gronichd, M. Yuklad, M.D. Fejgina,c, M. Lishnerc,d a
Genetic Institute, Meir Hospital, Kfar-Saba 44281, Israel b Bar Ilan University, Ramat-Gan, Israel c Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel d Department of Medicine and Hematology, Sapir Medical Center, Kfar-Saba, Israel Received 21 October 2005; received in revised form 28 November 2005; accepted 29 November 2005
Abstract
B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia of adults in Western countries. The most frequent recurring chromosomal aberrations identified in B-CLL patients are trisomy 12 and deletions of 13q, 17p, and 11q. Cases with deletions of 11q and 17p have a poor prognosis, whereas cases with deletions in 13q have a favorable prognosis. It was previously shown that CLL patients with trisomy 12 and del(13)(q14) have a higher rate of asynchronous replication of normal structural genes when compared to those with normal karyotypes. We studied the replication pattern of the structural locus 21q22 and the imprinted gene SNRPN and its telomere (15qter) and the random aneuploidy of chromosomes 9 and 18 in CLL patients with trisomy 12 and deletions of 11q and 17p, and compared the results to those of CLL patients without these aberrations and to healthy controls. Random aneuploidy rate was higher in the group of patients with trisomy 12 as compared to all other groups. The replication pattern with higher asynchronous pattern was found in both aberration groups compared to the CLL patients without the aberrations and to the control group with involvement of 21q22 and 15qter, whereas the highest synchronous group was found in the 2 aberrations CLL patient groups compared to the other groups with the imprinted locus SNRPN. The existence and significance of chromosomal aberrations in CLL have a deleterious effect on the processes of cell cycle and gene replication and may have biological and prognostic implications. Ó 2006 Elsevier Inc. All rights reserved.
1. Introduction B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia of adult populations in Western countries [1]. The morphology reveals a monomorphic small round cell population of B-lymphocytes. The disease tends to affect older patients, with a median age of 65 years usually cited [2]. The gender ratio varies from approximately 1.5 to 2.1 males to females [2]. CLL carries a variable prognosis, which is associated with various parameters including genetic aberrations. Classical cytogenetic analyses of bone marrow cells of BCLL patients are often hampered by low mitotic activity of malignant B-cells in culture, and normal chromosome results are obtained in the most patients. Molecular cytogenetic methods, especially fluorescence in situ hybridization
* Corresponding author: Tel.: 1972-9-737-2220; fax: 1972-9-7471296. E-mail address: [email protected] (A. Amiel). 0165-4608/06/$ – see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2005.11.019
(FISH), have enlarged the spectrum of genomic aberrations detected in B-CLL [3]. The most frequent recurring chromosomal aberrations identified in B-CLL patients are trisomy 12 and deletions of 13q, 17p, and 11q [3,4]. Patients with deletion 13q usually have a favorable prognosis and longer survival time, but patients with del(17p) display treatment failure in comparison to patients with del(11)(q22~q23) and trisomy 12 [3]. Deletion (11)(q22~q23) is characteristic of patients with poor prognosis [1,3,4]. The aneuploidy-cancer theory proposes that cancer is caused by the abnormal dosage of thousands of normal genes. This abnormal dosage of genes is generated by the gain or loss of specific chromosomes or segments of chromosomes (alias aneuploidy) [5]. However, the state of aneuploidy destabilizes chromosomes and genes because it unbalances highly conserved teams of proteins that segregate, synthesize, and repair chromosomes. Thus, the inherent instability of aneuploidy catalyzes a chain reaction of chromosome reassortments and rearrangements [5–9]. The theory predicts that chromosomal and genetic
146
A. Amiel et al. / Cancer Genetics and Cytogenetics 167 (2006) 145–149
instability is proportional to the degree of aneuploidy and to the types of chromosomes that are unbalanced; this was also confirmed by Fabarius et al. [10]. Numerous studies have demonstrated close association between the replication timing of a given chromosome region during S-phase and transcriptional activity of genes in this region. Hence, expressed loci replicate early in Sphase while silent loci replicate late [11–14]. By using this technique in human somatic cells, it has been convincingly demonstrated that alleles of genes which exhibit allelespecific expression (mono-allelic mode of expression), such as imprinted genes [15], genes subjected to X-chromosome inactivation [16] and olfactory receptor genes [17] replicate asynchronously, whereas alleles which are expressed concomitantly (biallelic mode of expression) replicate highly synchronously [14,18–20]. There is also evidence that alleles of loci, which normally replicate synchronously such as those associated with cell proliferation (TP53, HER2/ neu, and C-MYC ) or chromosome segregation, replicate asynchronously when present in cancer cells [19,21–23]. Some suggest that these alleles behave like the heterochromatic region and replicate in the late S-phase, whereas other believe that they replicate in the early S-phase or randomly during all the S-phase [24–26]. The aim of this study was to analyze whether CLL patients with different cytogenetic aberrations also differ in the parameter of spontaneous aneuploidy. In addition, we assessed the replication status of the CLL patients with and without the examined aberrations, with imprinted locus SNRPN and telomere 15qter, and the 21q22 locus.
2. Materials and methods The study was approved by the local ethical committee. Leukocytes from 54 CLL patients aged 49–88 years at both diagnosis and during treatment and followed at the Hematology Unit in Meir Medical Center were studied and compared to 7 healthy controls of matched age. The diagnosis of CLL was based on lymphocytosis greater than 5x106/mL and immunophenotyping that confirmed the monoclonal origin of the B-lymphocytes. Peripheral blood samples were obtained and incubated for short-term culture in an F10-supplemented medium in a 37 C moist chamber for 72 hours. The supplemented medium contained 20% FCS, lipopolysaccharide (40 mg/mL, 10 units/mL heparin, and 1% antibiotics. The leukocytes from the healthy controls were incubated in the F-10 supplemented medium contained PHA instead of LPS for 72 hours. After incubation, colchicine (final concentration 0.1 mg/mL) was added to the cultures for 1 hour, followed by hypotonic treatment (0.075 M KCl and 37 C for 15 minutes) and 4 washes, each with a fresh cold 3:1 methanolacetic acid solution. The lymphocyte suspensions of the three samples were stored at –4 C.
2.1. Probes For the random aneuploidy the Abbott Vysis probe, CEP 18 spectrum green (Cat No. 5J10-18) and CEPg spectrum orange (Cat No. 6J36-09). For the aberration detection, the following Abbot Vysis probes were used: CEP 12 spectrum (Cat No. 7J20-12); LSI ATM (ataxia telangiectasia mutation at 11q22.3) SpectrumOrange (Cat No. 5J6401); and LSI p53 (17p13.1) SpectrumOrange (Cat No. 5J52-01). For the replication pattern, the following probes were used: SNRPN (Small Nuclear Ribonucleoprotein Polypeptide N) imprinting center - Red Fluorophore with 15qter control probe green fluorophore (Cat No. LPU005, cytocell); and the Abbott Vysis probe 21q22 (Cat No. 8-5JB13-02). 2.2. Specimen pretreatment prior to co-denaturation The pretreatment procedure is highly recommended by Abbott/Vysis protocol prior to hybridization of ToTelVysion probes on lymphocyte specimens when using co-denaturation. The purpose of this procedure is to make the chromosomal DNA accessible for hybridization and to protect the morphology of the chromosomes from the co-denaturation process. 2.3. FISH technique Fresh slide preads were denatured for 2 minutes in 70% formamide standard saline citrate (2SSC) at 70 C and dehydrated in a graded ethanol series. The probe mix was then applied to air-warmed slides (30 mL, mix sealed under a 24x50 mm glass cover slip) and hybridized from 18 hours at 37 C in moist chamber. Following hybridization, the slides were washed in 50% formamide 2XSSC for 20 minutes at 43 C, rinsed in two changes of 2XSSC at 37 C for 4 minutes each, and placed in 0.05% Tween 20 (Sigma, Rehovot, Israel). For FISH analysis, the slides were counterstained in 4,6 diamidino-z-phenyindole (Sigma) antifade solution and analyzed for simultaneous viewing of FITC (Fluorescein Isothiocyanate), Texas Red, and 4,6 diamidinoz-phenyindole with an imaging processing system (Applied Imaging, Santa Clara, Ca.). 2.4. Cytogenetic evaluation Probes for chromosomes 9 and 18 were used for the detection of aneuploidy. For each cell, we recorded the number of hybridization signals. The rate of aneuploidy was inferred from the fraction of cells revealing 1, 3, or more hybridization signals per cell. For the replication pattern interphase cells with 2 hybridization signals were analyzed for each probe. The cells were classified into three categories according to Selig et al. [14]. The samples were scored ‘‘blindly’’. The level of synchrony in replication timing was derived from the fraction of SD cells.
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2.5. Statistical analysis Two 2-sample t-tests were applied for testing differences between the study groups for quantitative parameters. All tests were two-tailed and a p-value of 0.05 or less was considered statistically significant. We used Microsoft Excel software. 3. Results For the detection of the various aberrations, FISH with CEP12, LS1p53 (17p13.1), and the ATM locus (11q22.3) was performed. Ten (18.5%) out of the 54 CLL patients had trisomy 12, seven (13%) had deletion at TP53, and three (5.5%) had a deletion at the ATM locus. Two patients had both trisomy 12 and 17p deletion. One patient carried 2 deletions: at 17p and 11q. Because of the low number of individuals with the 11q and 17p deletions, we combined them into one group. We found higher aneuploidy rate (monosomy, trisomy, and tetrasomy) in the CLL study group compared to control group ( p ! 0.01; Tables 1 and 2). In the CLL group with normal FISH findings, trisomy and tetrasomy were significantly lower than the trisomy 12 study group with both chromosome analyzed ( p ! 0.05; Tables 1 and 2). Also the rate of monosomy, trisomy, and Table 1 Frequency of aneuploidy with chromosomes 9 and 18 in the control group(1), CLL patient without diagnosed chromosomal aberrations(2), CLL patients with trisomy 12(3) and CLL patients with a deletions of 17p and/or 11q(4) Average 6 standard deviation (%)
Number of samples Chromosome 9 1 8
2
10
3
9
4
8
Chromosome 18 1 8
2
10
3
9
4
8
Monosomy Disomy Trisomy and Monosomy Disomy Trisomy and Monosomy Disomy Trisomy and Monosomy Disomy Trisomy and Monosomy Disomy Trisomy and Monosomy Disomy Trisomy and Monosomy Disomy Trisomy and Monosomy Disomy Trisomy and
Tetrasomy
Tetrasomy
Tetrasomy
Tetrasomy
Tetrasomy
Tetrasomy
Tetrasomy
Tetrasomy
2.62 6 1.19 97.33 6 1.16 0.17 6 0.36 9.89 6 2.85 87.21 6 3.36 2.84 6 1.66 15.54 6 3.58 79.08 6 2.72 4.90 6 2.33 18.27 6 3.88 77.41 6 3.98 4.28 6 1.88 3 6 1.48 97.20 6 1.53 0.17 6 0.31 8.87 6 2.54 87.86 6 2.99 3.21 6 2.09 14.33 6 4.13 79.28 6 3.29 6.35 6 2.36 19.87 6 3.40 76.34 6 2.66 3.75 6 1.59
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Table 2 Statistical analyses of the average of aneuploidy in chromosomes 9 and 18 between all the different groups: (1) Control group, (2) CLL patients without of the chromosomal aberration, (3) CLL patients with trisomy 12, (4) CLL patient with a deletions of 17p and/or 11q. The numbers in the tables represent the p value of two tail-t-test Comparison between the experimental groups Chromosome 9 group (2) compared group (3) compared group (4) compared group (2) compared group (2) compared group (3) compared Chromosome 18 group (2) compared group (3) compared group (4) compared group (2) compared group (2) compared group (3) compared
Trisomy and Monosomy Disomy Tetrasomy
to to to to to to
group group group group group group
(1) (1) (1) (3) (4) (4)
0 0 0 0.0018 0.0002 0.1546
0 0 0 0 0.0001 0.3406
0.0006 0.0003 0.0004 0.0449 0.112 0.5511
to to to to to to
group group group group group group
(1) (1) (1) (3) (4) (4)
0 0 0 0.0045 0 0.0085
0 0 0 0 0 0.0598
0.0012 0 0 0.0075 0.541 0.0176
tetrasomy with chromosome 18 was significantly lower in the group with both deletions (11q and 17p) compared to the group with trisomy 12 ( p ! 0.01; Tables 1 and 2). The synchronous replication patterns of the 21q22 and 15qter were changed to asynchronous pattern (SD) on behalf of the SS pattern. In all CLL patients compared to control group the SD pattern was significantly higher. In the patients with abnormal FISH the rate of asynchrony was significantly higher then in those with normal FISH (Fig. 1a-c, Table 2). When the imprinted locus SNRPN was applied, a trend from asynchronous to synchronous replication in the CLL patients compared to controls was found, whereas in both aberrations groups the synchronous pattern was higher than the CLL group without aberrations ( p ! 0.01; Fig. 1b, Table 2). In all CLL patients, SS pattern was increased on behalf of the SD group (with the SNRPN locus), which means that one allele replicated later. We also found that the 15qter subtelomeric region was modifying the high synchronization pattern in the normal control group, to a synchronization pattern in all CLL patient group, and was the same in the groups with and without the aberrations (Table 2). The asynchrony rate (SD) was increased on behalf of the DD pattern, which means that one allele is replicating later on (Fig. 1c).
4. Discussion The random aneuploidy rate was significantly higher in patients with trisomy 12 compared to the patients with the deletions 11q and 17p and to CLL patients without aberrations. In addition, patients with a 17p deletion have the poorest outcome, followed by 11q deletion and trisomy 12 (that goes with the survival rate), it is possible that trisomy 12, which is a more serious aberration than a local deletion
A. Amiel et al. / Cancer Genetics and Cytogenetics 167 (2006) 145–149
148
a
Locus 21q22.
SD DD SS
CLL with a deletion of 17p or 11q
b
CLL with trisomy 12
CLL without chromosomal aberration
cell frequency (%)
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% control group
Locus SNRPN 100% 90% 70% 60%
SD DD SS
50% 40% 30% 20%
cell frequency (%)
80%
10% 0% CLL with a deletion of 17p or 11q
c
CLL with trisomy 12
CLL without aberration
control group
Locus 15qter.
SD DD SS
CLL with a deletion of 17p or 11q
CLL with trisomy 12
CLL without aberration
cell frequency (%)
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% control group
Fig. 1. Replication pattern of CLL patients and control group with the different loci analyzed.
has more effect on the nearest environment (cells) on the cell cycle progression and thus on chromosome segregation than on the disease phenotype. On the other hand nonexpression of specific loci (TP53 and ATM ) have more serious effect on the disease phenotype and progression [1,3,4]. In a study of gene expression a majority of the significant changes in gene expression were associated with the 11q23 deletion. Hierarchical clustering of statistically significant genes confirmed the similarity in gene expression among cases with the 11q23 deletion and pooled B-cell malignant cell lines. All the other CLL samples with 17p13 deletion, trisomy 12, 13q14 deletion, or normal karyotype were distributed randomly according to gene expression. Therefore, it may be concluded that certain chromosome abnormalities, such as the 11q23 deletion, may not only be markers of poor prognosis, but through their genetic lesion, drive tumor progression by a distinct transcriptional pathway [4]. The asynchronization rate was significantly different between the CLL patients without the examined aberrations
and both groups of aberrations with no difference between the two aberration groups, as was found before [27]. The existence of any of these aberrations has an unfavorable effect on the process of normal gene replication [27]. According to our results, it seems that the mechanisms of cell cycle progression and gene replication control are segregated with different sensitivity to the specific aberration. We found that the 15qter locus is a normal structural behaving locus, which replicates normally in a bi-allelic manner. In CLL patients, the mode of replication resembles the asynchronous pattern groups, similar to the normal structural locus 21q22. It seems that more than one mechanism is involved in the replicating control, which brings about the change from synchronous to asynchronous pattern in cancer cells. While in the 21q22 locus one of the alleles replicated earlier than anticipated in these patients, in the 15qter locus one allele replicated later than anticipated in the same group of patients. The imprinted gene SNPRN, which in normal control cells replicates in non-Mendelian manner (asynchronous) [28,29], changed the pattern of replication
A. Amiel et al. / Cancer Genetics and Cytogenetics 167 (2006) 145–149
towards synchronous manner in CLL patients, a change that had been previously described in cancer patients [28,29]. In both SNRPN and the sub-telomeric group 15qter (both on chromosome 15) one allele replicated later (the same mechanism). It is not clear whether this happened randomly or whether in this condition the subtelomeric loci mimic the behavior of the other loci on the same chromosome. Additional studies of the replication pattern of the different subtelomeric region are needed in order to find whether they share a common mechanism of replication mode in normal and cancer cells. In summary, the confirmed chromosomal aberrations in CLL disease have a deleterious and differential effect on cell cycle and gene replication processes and may have biological and prognostic implications. References [1] Goorha S, Glenn MJ, Drozd-Borysiuk E, Chen Z. A set of commercially available fluorescent in-situ hybridization probes efficient detects cytogenetic abnormalities in patients with chronic lymphocytic leukemia. Genet Med 2004;6:48–53. [2] Glassman AB, Hayes KJ. The value of fluorescence in situ hybridization in the diagnosis and prognosis of chronic lymphocytic leukemia. Cancer Genet Cytogenet 2005;158:88–91. [3] Sindelarova L, Michalova K, Zemanova Z, Ransdorfova S, Brezinova J, Pekova S, Schwarz J, Karban J, Cmunt E. Incidence of chromosomal anomalies detected with FISH and their clinical correlations in B-chronic lymphocytic leukemia. Cancer Genet Cytogenet 2005;160:27–34. [4] Dickinson JD, Smith LM, Sanger WG, Zhou G, Townley P, Lynch JC, Pavletic S, Bierman PJ, Joshi SS. Unique gene expression and clinical characteristics are associated with the 11q23 deletion in chronic lymphocytic leukemia. Brit J Haematol 2005;128:460–71. [5] Duesberg P, Rasnick D. Aneuploidy, the somatic mutation that makes cancer a species of its own. Cell Motil Cytoskel 2000;47:81–107. [6] Li R, Yerganian G, Duesberg P, Kraemer A, Willer A, Rausch C, Hechtmann R. Aneuploidy correlated 100% with chemical transformation of Chinese hamster cells. Proc Natl Acad Sci USA 1997; 94:14506–11. [7] Brinkley BR, Goepfert TM. Supernumerary centrosomes and cancer: Boveri’s hypothesis resurrected. Cell Motil Cytoskeleton 1998;41: 281–8. [8] Duesberg P, Rausch C, Rasnick D, Hehlmann R. Genetic instability of cancer cells is proportional to their degree of aneuploidy. Proc Natl Acad Sci USA 1998;95:13692–7. [9] Rasnick D, Duesberg P. How aneuploidy affects metabolic control and causes cancer. Biochem J 1993;340:621–30. [10] Fabarius A, Willer A, Yerganian G, Hehlmann R, Duesberg P. Specific aneusomies in Chinese hamster cell at different stages of neoplastic transformation, initiated by nitrosomethylurea. Proc Natl Acad Sci USA 2002;99:6773–83. [11] Goldman MA, Holmquist GP, Gray MC, Caston LA, Nag A. Replication timing of mammalian genes and middle repetitive sequences. Science 1984;224:686–92.
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