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In situ hybridization: A simple and sensitive method for detection of trisomy 12 in chronic lymphocytic leukemia
In Situ Hybridization: A Simple and Sensitive Method for Detection of Trisomy 12 in Chronic Lymphocytic Leukemia Arie Lalkin, Michael Lishner, Elena Gaber, Yosef Manor, Moshe Fejgin, Mordchai Ravid, and Aliza Amiel
ABSTRACT: Chromosome aberrations are detected in only 50% of patients with chronic lymphocytic leukemia (CLL), owing usually to the low mitotic rate exhibited by the neoplastic lymphocytes. Fluorescence in situ hybridization (FISH) is o simple method for identifying numerical abnormalities of the target chromosome in interphase nuclei. Therefore, we used the FISH procedure with chromosome 12-specific a-satellite probe to evaluate 19 patients with CLL. Trisomy 12 was detected in interphase cells of 12 patients (63%). Cytogenetic analysis, performed in nine patients, yielded trisomy 12 in four (44%). FISH detected three patients with trisomy 12 in w h o m conventional cytogenetic method yielded a normal karyotype. FISH is a simple, reliable, and sensitive method for detection of trisomy 12 in patients with CLL.
INTRODUCTION
PATIENTS AND METHODS
Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. The disease stems from a clonal proliferation and accumulation of neoplastic B cells [1]. Chromosomal aberrations are detected in 50% of patients [2-5], but conventional cytogenetic analysis is problematic either because analyzable metaphases are not obtained or a normal karyotype is noted in metaphases obtained [6]. In most cases with normal karyotypes, the metaphase cells studied were normal T cells [7]; in only a few cases were they clonal B cells with normal chromosomes [8]. Trisomy 12 is the most common chromosome abnormality in CLL [4, 9, 10]. Acquisition of an extra copy of chromosome 12 has been suggested to play a role in the pathogenesis of the disease [2, 11, 12]. In addition, trisomy 12 is an independent unfavorable prognostic indicator in CLL [2, 13, 14]. Fluorescence in situ hybridization (FISH) is a simple and rapid method for detection of specific DNA target sequences in metaphase chromosomes and in interphase nuclei [15]. Patients with CLL and normal karyotype may actually be trisomic in interphase cells, as was demonstrated by use of FISH technique with a chromosome 12-specific ¢z-satellite DNA probe [15]. Therefore, we further explored the potential of FISH in detection of trisomy 12 in CLL and compared it with conventional cytogenetic methods.
Nineteen consecutive patients with B-CLL were analyzed. The diagnosis was confirmed when patients were admitted to the hospital with lymphocytosis greater than 5 x 109/L and with immunologic studies that confirmed the monoclonal origin of the B lymphocytes. Data were collected with specific attention to clinical characteristics such as age, sex, clinical stage, previous and present treatment, and current blood counts.
From the Genetic Unit (E. G., M. F., A. A.), Hematology Unit (M. L., Y. M.), and Department of Medicine (,4.. L., M. R. ), Meir Hospital, Kfar Saba, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. Address reprint requests to: A. Amiel, Ph.D., Genetic Unit, Meir Hospital, Kfar-Saba 44281, Israel. Received January 14, •993; accepted May 11, 1993.
Cytogenetic Studies Peripheral blood (PB) [2 x 106/ml) was incubated in RPMI 1640 medium with 20% fetal calf serum. Two mitogens were used in all patients: lipopolysaccharide (LPS 40 ~tg/ml) and phytohemagglutinin (PHA Welcome, 0.1 mg/ml). Cells were harvested after 3-4 days according to standard procedures [16-16]. At least 10 metaphases but usually more than 20 were obtained from stimulated cultures and were karyotyped without knowledge of the FISH results (when performed). Unstained slides for FISH were stored at - 2O°C until the time of hybridization. Fresh or frozen slides spreads were treated with RNAse (100 ttg/ml) at 37°C for I hour. The slide preparations were then denatured for 2 minutes in 70% formamide/2 × SSC at 70°C and dehydrated in a graded ethanol series. The probe mix was then applied to air-warmed slides (30 ~tl mix sealed under a 24 x 50-mm glass coverslip) and hybridized for 18 hours at 37°C in a moist chamber. After hybridization, the slides were washed in 50% formamide/2 × SSC for 20 minutes at 43°C, rinsed in two changes of 2 x SSC at 37°C for 4 minutes each time, and placed in 0.05% Tween 40 (Sigma Chemical). Fluorescent detection was performed by alternating layers of FITC-avidin DCS (5 21
© 1993 Elsevier Science P u b l i s h i n g Co., Inc. 655 A v e n u e of the Americas, N e w York, NY 10010
Cancer Genet Cytogenet 70:21-24 (1993) 0165-4608/93/$06.00
22
A. Lalkin et al. RESULTS
A
Nine women and 10 men with a mean age of 72.5 + 14 years were studied. Four patients were in stage 0 (Rai classification), one was in stage I, six were in stage II, five were in stage III, and two were in stage IV (one was in unknown stage). Analysis of three control cases (Table 1, group b) demonstrated three signals in 1.3, 1.3, and 2.8 (mean 1.8%) of cells. These results are in accord with those of previous reports [16-18]. Twelve (63%) of the patients were trisomic for chromosome 12 (Table 1, group A). Cytogenetic analysis was performed in nine patients; in four (44%), trisomy 12 was detected. In these four patients, the trisomy was also demonstrated by FISH technique. In three patients with a normal karyotype, however, the trisomy was detected by FISH. The correlation between these techniques is shown in Table 1. Ten other patients were studied by FISH technique alone. Trisomy 12 was detected in five. No correlation was noted between age, stage, or previous treatment and the presence of trisomy 12. Similarly, there was no correlation between duration of disease and detection of trisomy or its frequency.
DISCUSSION
B Figure I Fluorescence in situ hybridization (FISH} studies with a chromosome 12-specific a-satellite DNA probe in patients with B-cell chronic lymphocytic leukemia. (A) Two interphase nuclei with two copies of chromosome 12 (two fluorescent signals). (B) Interphase nucleus with three copies of chromosome 12 (three fluorescent signals).
~tg/ml) and anti-avidin antibody (Vector Laboratories) until two layers of avidin had been applied. Each layer was incubated for 30 minutes at 37°C under a plastic coverslip in a moist chamber. Slides were washed in 0.05% Tween 40 buffer at room temperature between applications. Preparations were counterstained in a propidium iodide (PI)/antifade solution and analyzed by an Olympus BHS fluorescent microscope equipped with an FITC filter [450-490 ram). Two fluorescent spots showed the normal two copies of chromosome 12 (Fig. 1A and 1B), whereas three spots indicated a trisomic cell (Fig. 1B). We also counted cells with four or more fluorescent spots (Table 1). The presence of trisomy 12 was analyzed by a confidence interval (0.95) under the binomial distribution test (if the confidence interval was 0 or less, it was defined as an occasional finding). Correlation between clinical parameters and the incidence of trisomy was evaluated by Spirman correlation test.
Nine patients with CLL were evaluated by both conventional cytogenetic analysis and FISH with chromosome 12-specific a-satellite probe, and three patients with a normal karyotype demonstrated trisomy 12 by FISH. Ten other patients were studied by FISH alone, and three signals were detected in five of them. These results compare favorably with those of previous reports. These studies identified the trisomy by FISH in two of 13 [19], two of five [20], five of 30 [21], and four of 20 [15] patients with either a normal karyotype, inadequate chromosomes for cytogenetic analysis, or abnormal karyotype without trisomy 12. These studies suggest that FISH has an advantage in the range of 20-30% in detection of trisomy 12. Thus, our results and those of other studies indicate that FISH with a probe to chromosome 12 is a more sensitive method to detect trisomy 12 than is conventional cytogenetic analysis. This advantage of FISH is especially important in CLL, in which neoplastic B cells often fail to proliferate despite polyclonal B-cell activators; therefore, no metaphases are obtained [22, 23]. FISH enables evaluation of both metaphase and interphase cells [15, 19-21]. In addition, FISH is a rapid technique that enables analysis of single cells alone or in combination with immunophenotyping, study of archival slides, and scoring of more cells than are usually available in metaphase cytogenetics [21]. Because we used only a specific probe for chromosome 12, however, other chromosome aberrations, if present, were missed. Therefore, FISH should be used as a complement to classic karyotype analysis. Trisomy 12 has been suggested to be an unfavorable prognostic indicator in CLL [2, 14]. Furthermore, Anastasi et al. [21] reported that the impact of trisomy 12, analyzed retrospectively as detected by interphase analysis, was greater than that of conventional cytogenetics in prediction of poor survival. In our patients, there was no correlation between stage of CLL and presence of trisomy 12 such as was noted
In Situ Hybridization in B-CLL
23
Table 1 C h r o m o s o m e analysis and FISH in CLL patients and normal controls No. of signals Case/age (yr) Group A (CLL patients) 1/60 2/64 3/80 4/79 5/86 6/73 7/84 8/75 9/-10/78 11/61 12/81 13/75 14/80 15/72 16/62 17/80 18/58 19/58 Group B (healthy controls) 1/30 2/40 3/35
2
3
4 and more
Trisomy 12
110 61 120 120 46 62 130 135 105 120 133 190 215 135 130 72 69 162 100
14 18 19 35 4 0 13 12 4 0 6 11 1 22 3 7 11 1 10
0 0 0 0 0 O 0 1 0 0 1 2 0 0 0 0 2 0 0
+ + + + + + + + + + + +
47,XX, + 12,/45,XX, - 21 47,XY, + 12,del(3p) ND 47,XY, - 5, + 12t(2;9) ND 46,XY ND ND ND 46,XX ND 47,XX, + 12,t(12;14) ND 46,XY,del(2p) ND 46,XY ND ND 46,XY,del(9q)
150 145 103
2 2 3
0 0 0
-
46,XX 46,XY 46,XY
Cytogenetics
Abbreviations: FISH, fluorescence in situ hybridization; CLL, chronic lymphocytic leukemia; ND, not done.
in another study [12]. The small number of patients precludes conclusions regarding prognosis. Clearly, prospective studies are n e e d e d to clarify the significance of trisomy 12 detected by FISH as a prognostic factor. Trisomy 12 was reported to be the most frequent abnormal karyotype in patients w i t h CLL [4, 9, 10]. Han et al. [24] suggested that trisomy 12 is the primary karyotypic change in CLL and that other c h r o m o s o m e changes develop later as a result of clonal evolution. Based on the low i n c i d e n c e and m o s a i c i s m of this abnormality detected by nonradioactive ISH, Raghoebier et al. [25] c o n c l u d e d that trisomy 12 is not a primary event in leukemogenesis of CLL. T h e critical region involved in trisomy 12 appears to be in band q13-q21 on the long arm of c h r o m o s o m e 12 [26]. Bird et al. [6] suggested that genes located in this region provide CLL cell proliferative advantage w h e n they exist in triplicate. Few genes such as the homolog of the glioma-associated oncogene (GLI), the m u r i n e m a m m a r y t u m o r virus integration site (INT), or the ?-interferon gene are located in the critical region in c h r o m o s o m e 12, but none has been implicated in CLL [11]. Th e role of KRAS2 was also investigated, but no activation p o i n t mutation, gene amplification, or structural abnormalities were detected in the 48 patients studied [27]. FISH is a rapid a n d sensitive m e t h o d to detect trisomy 12 in patients w i t h CLL. Its m a i n advantage is that it allows study of interphase cells. It also enables evaluation of longstored slides and single cells. Prospective studies should
correlate FISH results w i t h the clinical course and prognosis and co m b i n e cytogenetic and m o l ecu l ar analysis of the critical region in c h r o m o s o m e 12 to evaluate the role of oncogenes and/or suppressor genes in the process of malignant transformation.
REFERENCES 1. Gale RP, Foon KA (1985): Chronic lymphocytic leukemia. Recent advances in biology and treatment. Ann Intern Med 103:101-120. 2. Juliusson G, Gahrton G (1990): Chromosome aberrations in B-cell chronic lymphocytic leukemia. Cancer Gent Cytogenet 45:143-160. 3. Fiona M, Stockdill RG (1987): Clonal chromosome abnormalities in chronic lymphocytic leukemia patients revealed by TPA stimulation of whole blood cultures. Cancer Genet Cytogenet 25:109-121. 4. Datta T, Bauchinger M, Emmerich B, Reichle A (1991): Chromosome analyses in chronic lymphocytic leukemia and related B-cell neoplasms. Cancer Genet Cytogenet 55:49-56. 5. Geisler CH, Phillip P, Hansen MM (1989): B-cell chronic lymphocytic leukaemia: Clonal chromosome abnormalities and prognosis in 89 cases. Eur J Haematol 43:397-403. 6. Bird ML, Ueshima Y, Rowley JD, Haren JM, Vardiman JW (1989): Chromosome abnormalities in B-cell chronic lymphocytic leukemia and their clinical correlations. Leukemia 3:182-191. 7. Knuutila S, Elonen E, Teerenhovi L, Rossi L, Leskinen R,
24
8.
9.
10.
11. 12.
13.
14.
15.
16.
17.
A. Lalkin et al. Bloomfield CD, de la Chapelle A (1986): Trisomy 12 in B cells of patients with B-cell chronic lymphocytic leukemia. N Engl J Med 314:865-869. Autio K, Elonen E, Teerenhovi L, Knuutila S (1987): Cytogenetic and immunologic characterization of mitotic cells in chronic lymphocytic leukaemia. Eur J Haematol 39:289-298. Gahrton G, Robert KH (1982): Chromosomal aberrations in chronic B-cell lymphocytic leukemia. Cancer Genet Cytogenet 6:171-181. Gahrton F, Robert KH, Friberg K, et al. (1980): Nonrandom chromosomal aberrations in chronic lymphocytic leukemia revealed by polyclonal B-cell-mitogenstimulation. Blood 56:640-647. Crossen PE (1989): Cytogenetic and molecular changes in chronic B-cell leukemia. Cancer Genet Cytogenet 43:143-150. Jnliusson G, Robert KH, Ost A, Friberg K, Biberfeld P, Nilsson B, Zech L, Gahrton G (1985): Prognostic information from cytogenetic analysis in chronic B-lymphocytic leukemia and leukemic immunocytoma. Blood 65:134-141. Robert KH, Gahrton G, Friberg K, Zech L, Nils~on B (1982): Extra chromosome 12 and prognosis in chronic lymphocytic leukemia. Scand J Haematol 28:163-168. Gahrton G, Juliusson G (1988): Clinical implication of chromosomal aberrations in chronic B-lymphocytic leukemia cells. Nouv Rev Fr Hematol 30:389-392. Cuneo A, Wlodarska I, Sayed Aly M, Piva N, Carli MG, Fagioli F, Tallarico A, Pazzi I, Ferrari L, Cassiman JJ, Van den Berghe H, Castoldi GL (1992): Non-radioactive in situ hybridization for the detection and monitoring of trisomy 12 in B-cell chronic lymphocytic leukemia. Br J Haematol 81:192-196. Devilee P, Thiery RF, Kievits T, Kolluri R, Hopman AHN, Willard HF, Pearson PL, Cornelisse CJ (1988): Detection of chromosome aneuploidy in interphase nuclei from human primary breast tumor using chromosome-specific repetitive DNA probes. Cancer Res 48:5825-5827. Hopman AHN, Ramaekers FCS, Raap AK, Beck JLM, Devilee P, van der Ploeg M, Vooius GP (1988): In situ hybridization as a tool to study numerical chromosome aberrations in solid bladder tumors. Histochemistry 89:307-316.
18. Nederlof PM, Van der Flier S, Raap AK, Tanke HJ, Van der Ploeg M, Kornips F, Geraedts JPM (1989): Detection of chromosome aberrations in interphase tumor nuclei by non-radioactive in situ hybridization. Cancer Genet Cytogenet 42:87-98. 19. Losacla AP, Wessman M, Tianen M, Hopman AHN, Willard HF, Sole F, Caballin MR, Woessner S, Knuutila S (1991): Trisomy 12 in chronic lymphocytic leukemia: An interphase cytogenetic study. Blood 78:775-779. 20. Qumsiyeh MB, Tharapel SA (1992): Interphase detection of trisomy 12 in B-cell chronic lymphocytic leukemia by fluorescence hybridization in situ. Leukemia 6:602-604. 21. Anastasi J, Le Beau MM, Vardiman JW, Fernald AA, Larson EA, Rowley JD (1992): Detection of trisomy 12 in chronic lymphocytic leukemia by fluorescence in situ hybridization to interphase cells: A simple and sensitive method. Blood 79: 1796-1801. 22. Gahrton G, Robert KH, Friberg K, Zech L, Bird AH (1980): Nonrandom chromosomal aberrations in chronic lymphocytic leukemia revealed by polyclonal B-cell-mitogenstimulation. Blood 56:640. 23. Autio K, Elonen E, Teerenhovi L, Knuutila S (1987): Cytogenetic and immunologic characterization of mitotic cells in chronic lymphocytic leukaemia. Eur J Haematol 39:289. 24. Han T, Henderson ES, Emrich LJ, Sandberg AA (1987): Prognostic significance of karyotypic abnormalitiesin B-cell chronic lymphocytic leukemia: An update. Semin Hemato124:257-263. 25. Raghoebier S, Kibbelaar RE, Kleiverda JK, Kluin-NelemansJC, Van Krieken JHJM, Kok F, Kluin Pb_M (1992): Mosaicism of trisomy 12 in chronic lymphocytic leukemia detected by nonradioactive in situ hybridization. Leukemia 6:1220-1226. 26. Gahrton G, Robert KH, Friberg K, Juliusson G, Biberfeld P, Zech L {1982): Cytogenetic mapping of the duplicated segment of chromosome 12 in lymphoproliferative disorders. Nature 297:513-514. 27. Browett PJ, Ganeshaguru K, Hoffbrand AV, Norton JD (1988): Absence of Kirsten-ras oncogene activation in B-cell chronic lymphocytic leukemia. Leuk Res 12:25-31.
ABSTRACT: Chromosome aberrations are detected in only 50% of patients with chronic lymphocytic leukemia (CLL), owing usually to the low mitotic rate exhibited by the neoplastic lymphocytes. Fluorescence in situ hybridization (FISH) is o simple method for identifying numerical abnormalities of the target chromosome in interphase nuclei. Therefore, we used the FISH procedure with chromosome 12-specific a-satellite probe to evaluate 19 patients with CLL. Trisomy 12 was detected in interphase cells of 12 patients (63%). Cytogenetic analysis, performed in nine patients, yielded trisomy 12 in four (44%). FISH detected three patients with trisomy 12 in w h o m conventional cytogenetic method yielded a normal karyotype. FISH is a simple, reliable, and sensitive method for detection of trisomy 12 in patients with CLL.
INTRODUCTION
PATIENTS AND METHODS
Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. The disease stems from a clonal proliferation and accumulation of neoplastic B cells [1]. Chromosomal aberrations are detected in 50% of patients [2-5], but conventional cytogenetic analysis is problematic either because analyzable metaphases are not obtained or a normal karyotype is noted in metaphases obtained [6]. In most cases with normal karyotypes, the metaphase cells studied were normal T cells [7]; in only a few cases were they clonal B cells with normal chromosomes [8]. Trisomy 12 is the most common chromosome abnormality in CLL [4, 9, 10]. Acquisition of an extra copy of chromosome 12 has been suggested to play a role in the pathogenesis of the disease [2, 11, 12]. In addition, trisomy 12 is an independent unfavorable prognostic indicator in CLL [2, 13, 14]. Fluorescence in situ hybridization (FISH) is a simple and rapid method for detection of specific DNA target sequences in metaphase chromosomes and in interphase nuclei [15]. Patients with CLL and normal karyotype may actually be trisomic in interphase cells, as was demonstrated by use of FISH technique with a chromosome 12-specific ¢z-satellite DNA probe [15]. Therefore, we further explored the potential of FISH in detection of trisomy 12 in CLL and compared it with conventional cytogenetic methods.
Nineteen consecutive patients with B-CLL were analyzed. The diagnosis was confirmed when patients were admitted to the hospital with lymphocytosis greater than 5 x 109/L and with immunologic studies that confirmed the monoclonal origin of the B lymphocytes. Data were collected with specific attention to clinical characteristics such as age, sex, clinical stage, previous and present treatment, and current blood counts.
From the Genetic Unit (E. G., M. F., A. A.), Hematology Unit (M. L., Y. M.), and Department of Medicine (,4.. L., M. R. ), Meir Hospital, Kfar Saba, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. Address reprint requests to: A. Amiel, Ph.D., Genetic Unit, Meir Hospital, Kfar-Saba 44281, Israel. Received January 14, •993; accepted May 11, 1993.
Cytogenetic Studies Peripheral blood (PB) [2 x 106/ml) was incubated in RPMI 1640 medium with 20% fetal calf serum. Two mitogens were used in all patients: lipopolysaccharide (LPS 40 ~tg/ml) and phytohemagglutinin (PHA Welcome, 0.1 mg/ml). Cells were harvested after 3-4 days according to standard procedures [16-16]. At least 10 metaphases but usually more than 20 were obtained from stimulated cultures and were karyotyped without knowledge of the FISH results (when performed). Unstained slides for FISH were stored at - 2O°C until the time of hybridization. Fresh or frozen slides spreads were treated with RNAse (100 ttg/ml) at 37°C for I hour. The slide preparations were then denatured for 2 minutes in 70% formamide/2 × SSC at 70°C and dehydrated in a graded ethanol series. The probe mix was then applied to air-warmed slides (30 ~tl mix sealed under a 24 x 50-mm glass coverslip) and hybridized for 18 hours at 37°C in a moist chamber. After hybridization, the slides were washed in 50% formamide/2 × SSC for 20 minutes at 43°C, rinsed in two changes of 2 x SSC at 37°C for 4 minutes each time, and placed in 0.05% Tween 40 (Sigma Chemical). Fluorescent detection was performed by alternating layers of FITC-avidin DCS (5 21
© 1993 Elsevier Science P u b l i s h i n g Co., Inc. 655 A v e n u e of the Americas, N e w York, NY 10010
Cancer Genet Cytogenet 70:21-24 (1993) 0165-4608/93/$06.00
22
A. Lalkin et al. RESULTS
A
Nine women and 10 men with a mean age of 72.5 + 14 years were studied. Four patients were in stage 0 (Rai classification), one was in stage I, six were in stage II, five were in stage III, and two were in stage IV (one was in unknown stage). Analysis of three control cases (Table 1, group b) demonstrated three signals in 1.3, 1.3, and 2.8 (mean 1.8%) of cells. These results are in accord with those of previous reports [16-18]. Twelve (63%) of the patients were trisomic for chromosome 12 (Table 1, group A). Cytogenetic analysis was performed in nine patients; in four (44%), trisomy 12 was detected. In these four patients, the trisomy was also demonstrated by FISH technique. In three patients with a normal karyotype, however, the trisomy was detected by FISH. The correlation between these techniques is shown in Table 1. Ten other patients were studied by FISH technique alone. Trisomy 12 was detected in five. No correlation was noted between age, stage, or previous treatment and the presence of trisomy 12. Similarly, there was no correlation between duration of disease and detection of trisomy or its frequency.
DISCUSSION
B Figure I Fluorescence in situ hybridization (FISH} studies with a chromosome 12-specific a-satellite DNA probe in patients with B-cell chronic lymphocytic leukemia. (A) Two interphase nuclei with two copies of chromosome 12 (two fluorescent signals). (B) Interphase nucleus with three copies of chromosome 12 (three fluorescent signals).
~tg/ml) and anti-avidin antibody (Vector Laboratories) until two layers of avidin had been applied. Each layer was incubated for 30 minutes at 37°C under a plastic coverslip in a moist chamber. Slides were washed in 0.05% Tween 40 buffer at room temperature between applications. Preparations were counterstained in a propidium iodide (PI)/antifade solution and analyzed by an Olympus BHS fluorescent microscope equipped with an FITC filter [450-490 ram). Two fluorescent spots showed the normal two copies of chromosome 12 (Fig. 1A and 1B), whereas three spots indicated a trisomic cell (Fig. 1B). We also counted cells with four or more fluorescent spots (Table 1). The presence of trisomy 12 was analyzed by a confidence interval (0.95) under the binomial distribution test (if the confidence interval was 0 or less, it was defined as an occasional finding). Correlation between clinical parameters and the incidence of trisomy was evaluated by Spirman correlation test.
Nine patients with CLL were evaluated by both conventional cytogenetic analysis and FISH with chromosome 12-specific a-satellite probe, and three patients with a normal karyotype demonstrated trisomy 12 by FISH. Ten other patients were studied by FISH alone, and three signals were detected in five of them. These results compare favorably with those of previous reports. These studies identified the trisomy by FISH in two of 13 [19], two of five [20], five of 30 [21], and four of 20 [15] patients with either a normal karyotype, inadequate chromosomes for cytogenetic analysis, or abnormal karyotype without trisomy 12. These studies suggest that FISH has an advantage in the range of 20-30% in detection of trisomy 12. Thus, our results and those of other studies indicate that FISH with a probe to chromosome 12 is a more sensitive method to detect trisomy 12 than is conventional cytogenetic analysis. This advantage of FISH is especially important in CLL, in which neoplastic B cells often fail to proliferate despite polyclonal B-cell activators; therefore, no metaphases are obtained [22, 23]. FISH enables evaluation of both metaphase and interphase cells [15, 19-21]. In addition, FISH is a rapid technique that enables analysis of single cells alone or in combination with immunophenotyping, study of archival slides, and scoring of more cells than are usually available in metaphase cytogenetics [21]. Because we used only a specific probe for chromosome 12, however, other chromosome aberrations, if present, were missed. Therefore, FISH should be used as a complement to classic karyotype analysis. Trisomy 12 has been suggested to be an unfavorable prognostic indicator in CLL [2, 14]. Furthermore, Anastasi et al. [21] reported that the impact of trisomy 12, analyzed retrospectively as detected by interphase analysis, was greater than that of conventional cytogenetics in prediction of poor survival. In our patients, there was no correlation between stage of CLL and presence of trisomy 12 such as was noted
In Situ Hybridization in B-CLL
23
Table 1 C h r o m o s o m e analysis and FISH in CLL patients and normal controls No. of signals Case/age (yr) Group A (CLL patients) 1/60 2/64 3/80 4/79 5/86 6/73 7/84 8/75 9/-10/78 11/61 12/81 13/75 14/80 15/72 16/62 17/80 18/58 19/58 Group B (healthy controls) 1/30 2/40 3/35
2
3
4 and more
Trisomy 12
110 61 120 120 46 62 130 135 105 120 133 190 215 135 130 72 69 162 100
14 18 19 35 4 0 13 12 4 0 6 11 1 22 3 7 11 1 10
0 0 0 0 0 O 0 1 0 0 1 2 0 0 0 0 2 0 0
+ + + + + + + + + + + +
47,XX, + 12,/45,XX, - 21 47,XY, + 12,del(3p) ND 47,XY, - 5, + 12t(2;9) ND 46,XY ND ND ND 46,XX ND 47,XX, + 12,t(12;14) ND 46,XY,del(2p) ND 46,XY ND ND 46,XY,del(9q)
150 145 103
2 2 3
0 0 0
-
46,XX 46,XY 46,XY
Cytogenetics
Abbreviations: FISH, fluorescence in situ hybridization; CLL, chronic lymphocytic leukemia; ND, not done.
in another study [12]. The small number of patients precludes conclusions regarding prognosis. Clearly, prospective studies are n e e d e d to clarify the significance of trisomy 12 detected by FISH as a prognostic factor. Trisomy 12 was reported to be the most frequent abnormal karyotype in patients w i t h CLL [4, 9, 10]. Han et al. [24] suggested that trisomy 12 is the primary karyotypic change in CLL and that other c h r o m o s o m e changes develop later as a result of clonal evolution. Based on the low i n c i d e n c e and m o s a i c i s m of this abnormality detected by nonradioactive ISH, Raghoebier et al. [25] c o n c l u d e d that trisomy 12 is not a primary event in leukemogenesis of CLL. T h e critical region involved in trisomy 12 appears to be in band q13-q21 on the long arm of c h r o m o s o m e 12 [26]. Bird et al. [6] suggested that genes located in this region provide CLL cell proliferative advantage w h e n they exist in triplicate. Few genes such as the homolog of the glioma-associated oncogene (GLI), the m u r i n e m a m m a r y t u m o r virus integration site (INT), or the ?-interferon gene are located in the critical region in c h r o m o s o m e 12, but none has been implicated in CLL [11]. Th e role of KRAS2 was also investigated, but no activation p o i n t mutation, gene amplification, or structural abnormalities were detected in the 48 patients studied [27]. FISH is a rapid a n d sensitive m e t h o d to detect trisomy 12 in patients w i t h CLL. Its m a i n advantage is that it allows study of interphase cells. It also enables evaluation of longstored slides and single cells. Prospective studies should
correlate FISH results w i t h the clinical course and prognosis and co m b i n e cytogenetic and m o l ecu l ar analysis of the critical region in c h r o m o s o m e 12 to evaluate the role of oncogenes and/or suppressor genes in the process of malignant transformation.
REFERENCES 1. Gale RP, Foon KA (1985): Chronic lymphocytic leukemia. Recent advances in biology and treatment. Ann Intern Med 103:101-120. 2. Juliusson G, Gahrton G (1990): Chromosome aberrations in B-cell chronic lymphocytic leukemia. Cancer Gent Cytogenet 45:143-160. 3. Fiona M, Stockdill RG (1987): Clonal chromosome abnormalities in chronic lymphocytic leukemia patients revealed by TPA stimulation of whole blood cultures. Cancer Genet Cytogenet 25:109-121. 4. Datta T, Bauchinger M, Emmerich B, Reichle A (1991): Chromosome analyses in chronic lymphocytic leukemia and related B-cell neoplasms. Cancer Genet Cytogenet 55:49-56. 5. Geisler CH, Phillip P, Hansen MM (1989): B-cell chronic lymphocytic leukaemia: Clonal chromosome abnormalities and prognosis in 89 cases. Eur J Haematol 43:397-403. 6. Bird ML, Ueshima Y, Rowley JD, Haren JM, Vardiman JW (1989): Chromosome abnormalities in B-cell chronic lymphocytic leukemia and their clinical correlations. Leukemia 3:182-191. 7. Knuutila S, Elonen E, Teerenhovi L, Rossi L, Leskinen R,
24
8.
9.
10.
11. 12.
13.
14.
15.
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