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Trisomy 12 in chronic lymphocytic leukemia
Trisomy 12 in Chronic Lymphocytic Leukemia An Interphase Cytogenetic Study by Fluorescence In Situ Hybridization Y. L. Kwong, J. Pang, L. M. Ching, H. W. Liu, R. H. S. Liang, and L. C. Chan
ABSTRACT: B-cell chronic lymphocytic leukemia {CLL)is a rare disorder in the Chinese population. We evaluated the use of fluorescence in situ hybridization (FISH) with a chromosome 12-specific probe in the detection of trisomy 12 in interphase ceils of 19 Chinese CLL patients. FISH successfully detected trisomy 12 in two cases, one of which had normal conventional c~ogenetic findings, giving an incidence of 10%. The low incidence of tr/somy 12 in our CLL patients may also reflect a biologic difference of this rare disorder in our population, compared to that of the West.
INTRODUCTION B-cell chronic lymphocytic leukemia (CLL}is the most common leukemia in the West but is rare in the Orient, comprising only about 2 % of all leukemias in the Japanese [1] and Singapore Chinese [2], which was only 1/10 of the reported incidence of 25% of all leukemias in the West [3]. It has been shown that cytogenetic abnormalities may be of prognostic significance In CLL [4-7]. Trisomy 12 is the most c o m m o n numerical aberration,and constitutedaround 3 0 % of cases successfullyanalyzed [8].Because of the relativelyhigh failureroteof conventional cytogenetic analysis in CLL, despite the use of mitogens [9],the use of fluorescence in situ hybridization has been shown to give a more accurate reflectionof the incidence of trisomy 12 in CLL [I0-13]. We conducted a retrospective study of 19 cases of Chinese patients with CLL, in order to define the incidence of trisomy 12 in this rare disorder in our population.
MATERIALS AND METHODS Patients Five hundred and thirty-five newly diagnosed leukemic patients were seen in Queen Mary Hospital, Hens Kong, between January 1987 and June 1992. Thirty-five [6.5%) ofthese patients fit the diagnosis of CLL, of whom 19 had cytogenetic
From the University Department of Medicine, and University Department of Pathology, Queen Mary Hospital, Pokfulam Bead,
Hong Kong Address reprint requests to: Dr. Y. L. Kwong, University Department of Medicine, Professorial Block, Queen Mary Hospital, Pokfulam Bead, Hong Kong. Received March 3, 1993; accepted July 15, 1993.
investigations and were included in this study. Staging was based on the system recommended by the International Workshop on CLL [14].
Conventional Cytogenetic Analysis Marrow cells at diagnosis were used for 3-day cell cultures. Two cultures were set up, in the presence of lipopolysaccharide {100 pg/ml; Sigma, St. Louis, MO) and 12-Otetradecanoylphorbol-13-acetate [TPA; 2 pg/ml; Sigma), respectively. Metaphase chromosomes were banded by trypsin/Giemsa and karyotyped according to ISCN 1991 [15]. In Situ Hybridization In situ hybridiT-qtion was performed by a biotin-labeled chromosome 12 specific a satellite DNA probe {D12Z3, Oncor, Gaithersburg, MD) on archival specimens, which had been stored in methanol:glacial acetic acid {3:1 vohvol) for up to 2 years [16]. For each patient, the interphase cells were derived from the same cultures as those of conventional cytogenetic Ans]ysis. A 3-day phytohem~g.~alufinin-stimulated lymphocyte culture of a normR1 donor and metaphase preparation of a case [patient 1, Table 1} with trisomy 12 found on conventional cytogeneticanalysisof TPA-stimulated marrow culture were used as normal and positivecontrols for each of the experiments. Immediately before hybridization, the cellswere denatured In a solution contRining 70% formamlde, 4 x standard saline citrate[SCC} at 70°C for 2 minutes, and seriallydehydrated.Probe {20 ng foreach slide} was denatured in 15 ~l hybridizationbuffercontaining 5 0 % formAmide, 10% dextran, and 2 x SSC at 70°C for 2 minutes. In situ hybridization was carried out at 37°C overnight In a moisture chamber. Post-hybridization washing was performed in 2 x SSC with 50% formamide for 40 minutes at 44°C. The slides were then blocked with 3% bovine serum albumin In 4 x SSC. One hundred microliters FITC-avidin 83
© 1994 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytosenet 72:83-85 (1994)
0165-4608/94/$07.00
84
Y.L. Kwong et al.
Table 1 Clinical, conventional cytogenetic, and FISH findings in 19 CLL patients In Situ Hybridization
Conventional cytogenetics Case
Sex/age
Stage
Karyotype
M/32 M/65 M/69 M/38 M/56 M/68 M/54 M/71 M/64 M/64 M/67 M/60
A(I) C(III) B(II) B(II) B(II) C(III) C(III) A(III) C(IV) C(HI) C(HI) C{III) B(II) CflII) B(H) A(O) B(II) B (II) C(IV)
Normal + 12 c Inadequate Normal Inadequate t(3;7) d Normal + 3e Normal Complexf Normal Normal Inadequate Normal
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
F/77 M/58 F/72 F/67 M/72 M/50 M/56
M174
ComplexS Complex h Normal t(14;15) i Inadequate Normal
% of cells with s i g n a l #
% of cells analyzed
0b
1
12% 80% 75% 100% 50% 100% 40% -
1 + 1 1.5 8 3 3 0 9 1.5 0 . 8 ± 0.2 6 17 1 0 4.5 9 0 0.5 8 9 4
7 ± 3 5.5 6 12 8 8.5 29 12 6.2 ± 1.3 4.3 16.5 6 4 7 15 9 2.5 6 12 8
2 9 1 . 4 ± 7.6 67 86 85 88 90.5 62 85.5 8 3 . 4 ± 7.3 88.3 66.5 92.7 95 88 76 90 95.5 86 78.3 78
3
4
0.5 ± 0 . 4 26 0 0 1 1 0 1 9.6 ± 1.2 1 0 0.3 1 0.5 0 1 1.5 0 0.7 0
0.1 0 0 0 0 0 0 0 0 0.5 0 0 0 0 0 0 0 0 0 0
No. of cells analyzed 800 400 100 100 100 200 10O 200 500 400 200 300 100 200 100 100 200 100
3OO 100
Abbreviation: C, control. Q When more than 1 experiment was done, results expressed as mean ± 2 standard deviations.
b Including cells with no or doubtful signals, c 47,XY, + 1213]. d 43-45,XY,t(3;7)[p10;q10)[5], - 1717], - 1815][cp8]. e 47,XY,
+
313].
f 39-45,XY, - 6,add(13)(ql2), - 18, + mar[cpl0]. s 44-46~XX~dd(~(p32~[2]~del~)(q2~[6]~der(7)t(~;~)(q2~;q22)[~]~inv(9~p12q2~)[6]~add(17)(p~2)[6] [cpS]. h 41-46,XX,der(4)t(4;?)(p16;?),del{14)(q24)[cp10]. i 46,XY,t(14;15)(q32;p13)[4].
(25 tLg/ml) was added and the slides were incubated in the dark at 37°C for 40 minutes. After washing, 100 ~1 of biotinylated anti-avidin antibody (2.5 ~g/ml) was added, incubated for 40 minutes, washed, and subjected to a further 40-minute incubation with 100 ~I FrrC-avidin. After the last washings the slideswere mounted in phosphate-buffered saline covtainlng propidium iodide {1 ~g/ml) and examined immediately for fluorescent hybridization signals under U V light. 100-800 consecutive unselected cells were scored. RESULTS Patients The clinical, laboratory, and conventional cytogenetic findings of the patients are stlmmAT'iZed in Table 1. Patient 1 had trisomy 12 on cytogenetic analysisand was used as the pos itive control in all experiments.
0 and 1 hybridization signals on five separate experiments comprising a ~ of 800 cells were 1% and 7%, respectively. For the patient materials, these frequencies were somewhat higher, at 4.5% and 9.3%, respectively, probably reflecting deteriorationduring storage.Three hybridizationsi~rmlRwere found in 0.5% of cells in the control, 0.3-1.5% of cells in 10 patients,and none in seven patients.These patientswere considered to be negative. Patient 1 showed trisomy 12 in 12% of nmtephases o nconventional cyt~enetic RnA!~is, and in 26% of cells on ISH. Patient 8 showed normal metaphases on cytogenetic AnRIysis, but 9.6% of cells with three un ~tuivocal hybridization signals on four separate FISH experiments comprising 500 cells. This patient was considered to have a clone of cells with trisomy 12. DISCUSSION T h e relative rarity of CLL at 7% o f all l e u k e m i a i n o u r C h i o
In Situ Hybridlufion The results of in situ hybridization are summarized in Table 1. I n the normal control, the mean frequencies of finding
nase population accounted for the small number of cases we could investigate. Despite this, our study still illustrates several interesting points. First,in situ hybridization (FISH) has a high specificity
Trisomy 12 in CLL
for detection of trisomy 12, the false positive rate {three signals} being less than 1% of analyzed cells in the control and 2% in the patient materials. H S H successfully identified one case with trisomy 12 on conventional cytogenetic analysis, and also one case with normal conventional cytogenetic findings. In previous studies of CLL with normal conventional cytogenetics but trisomy 12 on FISH, 5-59% of interphase cells showed three hybridization signals [10-13]. This variation was also seen in our study, which might be due to the different types of preparations used. In our patients, we had no access to uncultured cells or cells from short-term (24hour) cultures. It is thus conceivable that in patient 8 the 3-day culture conditions were not optimal, so that only 10% of CLL cells survived, accounting for the normal cytogenetics (probably reflecting dividing T cells) and the rather low percentage of trisomy 12 cells. We had included cases with abnormal cytogenetics in our analysis, as Anastasi et al. [11] had shown that trisomy 12 clones might coexist with clones bearing other karyotypic aberrations. However, this phenomenon was not found in our patients. The use of ISH, w h e n compared with conventional cytogenotic analysis, increased our diagnostic yield of trisomy 12 from 5% to 10%, a twofold increase comparable to results in other series [10-12]. Although the number of patients in this study was small, the overall incidence of 10% of trisomy 12 is low compared to that of around 30% in the West. Further studies of oriental CLL patients will be required to confirm whether this low incidence of trisomy 12 in our patients reflects one of the biological differences of this rare disorder in the East as compared to the West. The authors thank A. Y. Y. Chan end T. S. K. Wen for technical assistence in conventional cytogenetic analysis. REFERENCES 1. Weias NS {1978):Geographical varietion in the incidence of the leukemias end lymphomas. In: Second Symposium on Epidemielegy and Cancer Registries in the Pacific Basin, Henderson BE, ed: Natl Cancer Inst Monogr 53. N.I.H. Publication N. 79-1864. 2. Wells R, Lau KS {1960):Incidence of leukemia in Singapore and rarity of chronic lymphocytic leukemia in Chinese. Br Med J 1:759. 3. Rai KR, Sawitsky A {1985):Diagnosis and treatment of chronic lymphocytic leukemia. In: Neoplastic Diseases of the Blood.
85
4.
5. 6. 7.
8. 9.
10.
Wiernik PH, Canellos GP, Kyle RA, Schiffer CA, eds. Churchill Livingstone, New York, pp. 105-120. Hen T, Ozer H, Sadamori N, Emrich L, Gomez GA, Henderson ES, Bloom ML, Sandberg AA (1984): Prognostic importance of cytngenetic abnormalities in patients with chronic lymphocytic leukemia. N Engl J Med 310:288-292. Bird ML, Uoshima Y, Rowley JD, Haren JM, ~miiman JW (1989): Chromosome abnormalities in B cell chronic lymphocytic leukemia and their clinical correlations. Leukemia 3:182-191. Geislar CH, Philip P, Hansen MM (1989): B-cell chronic lymphocytic leukemia: Clonsl chromosome abnormalities and prognosis in 89 cases. Eur J Haematol 1989;43:397-403. Juliusson G, Oscier DG, Fitchett M, Ross FM, Stockdill G, Mackie IvfJ, Parker AC, Castoldi GL, Cuneo A, Knuutila S, Elonan E, Gharton G (1990): Prognostic subgroups in B-cell chronic lymphocytic leukemia defined by specific chromosomal abnormalities. N Engl J Med 323:720-724. Crossen PE (1989): Cytogenetic and molecular changes in chronic B-cell leukemia. Cancer Genet Cytogenet 43:143-150. Sadamori N, Matsui S, Hen T, Sandbarg AA (1984): Comparative results with various polyclonal B-cell activators in aneuploid chronic lymphocytic leukemia. Cancer Genet Cytogenet 11:25-29. Losada AP, Wassman M, Tiainen M, Hopman AHN, Willard HF, Sol~ F, Caballin MR, Woessnar S, Knuutila S (1991): Trisomy 12 in chronic lymphocytic leukemia: an interphase cytogenetic study. Blood 78:775-779.
11. Anastasi J,Le Beau M M , Wardiman JW, Fernald AA, Larson RA, Rowley JD {1992): Detection of trisomy 12 in chronic lymphocytic leukemia by fluorescence in situ hybridization to interphase cells:a simple and sensitivemethod. Blood 79:1796-1801. 12. Cuneo A, Wlodarska I, Aly MS, Piva N, Carli MG, Fagioli F, Tallarico A, Pazzi I, FerrariL, Cassiman JJ,Van Den Berghe H, Castoldi GL (1992}:Non-radioactive in situ hybridization forthe detection and monitoring of trisomy 12 in B-cell chronic lymphocytic leukaemia. Br J Haematol 81:192-196. 13. Qumsiyeh MB, Thampel SA (1992}: Interphase detection of trisomy 12 in B-cell chronic lymphocytic leukemia by fluorescence hybridization in situ. Leukemia 6:602-605. 14. International Workshop on Chronic Lymphocytic Leukemia (1989}: Chronic lymphocytic leukemia: Recommendations for diagnosis, staging, and response criteria. A n n Intern M e d 110:236-238. 15. ISCN {1991}: Guidelines Cancer Cytogenetics. Supplement to A n International System for H u m a n Cytogenetic Nomenclature. Mitelman F, ed. Kargar, Basel, 1991. 16. Price CM, Kanfer EJ, Colman SM, Wastwood N, Barrett AJ,
Greaves MF (1991): Simultaneous genotypic and immunophenotypic analysis of interphase cells using dual-color fluorescence: a demonstration of lineage involvement in polycythemia vera. Blood 80:1033-1038.
ABSTRACT: B-cell chronic lymphocytic leukemia {CLL)is a rare disorder in the Chinese population. We evaluated the use of fluorescence in situ hybridization (FISH) with a chromosome 12-specific probe in the detection of trisomy 12 in interphase ceils of 19 Chinese CLL patients. FISH successfully detected trisomy 12 in two cases, one of which had normal conventional c~ogenetic findings, giving an incidence of 10%. The low incidence of tr/somy 12 in our CLL patients may also reflect a biologic difference of this rare disorder in our population, compared to that of the West.
INTRODUCTION B-cell chronic lymphocytic leukemia (CLL}is the most common leukemia in the West but is rare in the Orient, comprising only about 2 % of all leukemias in the Japanese [1] and Singapore Chinese [2], which was only 1/10 of the reported incidence of 25% of all leukemias in the West [3]. It has been shown that cytogenetic abnormalities may be of prognostic significance In CLL [4-7]. Trisomy 12 is the most c o m m o n numerical aberration,and constitutedaround 3 0 % of cases successfullyanalyzed [8].Because of the relativelyhigh failureroteof conventional cytogenetic analysis in CLL, despite the use of mitogens [9],the use of fluorescence in situ hybridization has been shown to give a more accurate reflectionof the incidence of trisomy 12 in CLL [I0-13]. We conducted a retrospective study of 19 cases of Chinese patients with CLL, in order to define the incidence of trisomy 12 in this rare disorder in our population.
MATERIALS AND METHODS Patients Five hundred and thirty-five newly diagnosed leukemic patients were seen in Queen Mary Hospital, Hens Kong, between January 1987 and June 1992. Thirty-five [6.5%) ofthese patients fit the diagnosis of CLL, of whom 19 had cytogenetic
From the University Department of Medicine, and University Department of Pathology, Queen Mary Hospital, Pokfulam Bead,
Hong Kong Address reprint requests to: Dr. Y. L. Kwong, University Department of Medicine, Professorial Block, Queen Mary Hospital, Pokfulam Bead, Hong Kong. Received March 3, 1993; accepted July 15, 1993.
investigations and were included in this study. Staging was based on the system recommended by the International Workshop on CLL [14].
Conventional Cytogenetic Analysis Marrow cells at diagnosis were used for 3-day cell cultures. Two cultures were set up, in the presence of lipopolysaccharide {100 pg/ml; Sigma, St. Louis, MO) and 12-Otetradecanoylphorbol-13-acetate [TPA; 2 pg/ml; Sigma), respectively. Metaphase chromosomes were banded by trypsin/Giemsa and karyotyped according to ISCN 1991 [15]. In Situ Hybridization In situ hybridiT-qtion was performed by a biotin-labeled chromosome 12 specific a satellite DNA probe {D12Z3, Oncor, Gaithersburg, MD) on archival specimens, which had been stored in methanol:glacial acetic acid {3:1 vohvol) for up to 2 years [16]. For each patient, the interphase cells were derived from the same cultures as those of conventional cytogenetic Ans]ysis. A 3-day phytohem~g.~alufinin-stimulated lymphocyte culture of a normR1 donor and metaphase preparation of a case [patient 1, Table 1} with trisomy 12 found on conventional cytogeneticanalysisof TPA-stimulated marrow culture were used as normal and positivecontrols for each of the experiments. Immediately before hybridization, the cellswere denatured In a solution contRining 70% formamlde, 4 x standard saline citrate[SCC} at 70°C for 2 minutes, and seriallydehydrated.Probe {20 ng foreach slide} was denatured in 15 ~l hybridizationbuffercontaining 5 0 % formAmide, 10% dextran, and 2 x SSC at 70°C for 2 minutes. In situ hybridization was carried out at 37°C overnight In a moisture chamber. Post-hybridization washing was performed in 2 x SSC with 50% formamide for 40 minutes at 44°C. The slides were then blocked with 3% bovine serum albumin In 4 x SSC. One hundred microliters FITC-avidin 83
© 1994 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytosenet 72:83-85 (1994)
0165-4608/94/$07.00
84
Y.L. Kwong et al.
Table 1 Clinical, conventional cytogenetic, and FISH findings in 19 CLL patients In Situ Hybridization
Conventional cytogenetics Case
Sex/age
Stage
Karyotype
M/32 M/65 M/69 M/38 M/56 M/68 M/54 M/71 M/64 M/64 M/67 M/60
A(I) C(III) B(II) B(II) B(II) C(III) C(III) A(III) C(IV) C(HI) C(HI) C{III) B(II) CflII) B(H) A(O) B(II) B (II) C(IV)
Normal + 12 c Inadequate Normal Inadequate t(3;7) d Normal + 3e Normal Complexf Normal Normal Inadequate Normal
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
F/77 M/58 F/72 F/67 M/72 M/50 M/56
M174
ComplexS Complex h Normal t(14;15) i Inadequate Normal
% of cells with s i g n a l #
% of cells analyzed
0b
1
12% 80% 75% 100% 50% 100% 40% -
1 + 1 1.5 8 3 3 0 9 1.5 0 . 8 ± 0.2 6 17 1 0 4.5 9 0 0.5 8 9 4
7 ± 3 5.5 6 12 8 8.5 29 12 6.2 ± 1.3 4.3 16.5 6 4 7 15 9 2.5 6 12 8
2 9 1 . 4 ± 7.6 67 86 85 88 90.5 62 85.5 8 3 . 4 ± 7.3 88.3 66.5 92.7 95 88 76 90 95.5 86 78.3 78
3
4
0.5 ± 0 . 4 26 0 0 1 1 0 1 9.6 ± 1.2 1 0 0.3 1 0.5 0 1 1.5 0 0.7 0
0.1 0 0 0 0 0 0 0 0 0.5 0 0 0 0 0 0 0 0 0 0
No. of cells analyzed 800 400 100 100 100 200 10O 200 500 400 200 300 100 200 100 100 200 100
3OO 100
Abbreviation: C, control. Q When more than 1 experiment was done, results expressed as mean ± 2 standard deviations.
b Including cells with no or doubtful signals, c 47,XY, + 1213]. d 43-45,XY,t(3;7)[p10;q10)[5], - 1717], - 1815][cp8]. e 47,XY,
+
313].
f 39-45,XY, - 6,add(13)(ql2), - 18, + mar[cpl0]. s 44-46~XX~dd(~(p32~[2]~del~)(q2~[6]~der(7)t(~;~)(q2~;q22)[~]~inv(9~p12q2~)[6]~add(17)(p~2)[6] [cpS]. h 41-46,XX,der(4)t(4;?)(p16;?),del{14)(q24)[cp10]. i 46,XY,t(14;15)(q32;p13)[4].
(25 tLg/ml) was added and the slides were incubated in the dark at 37°C for 40 minutes. After washing, 100 ~1 of biotinylated anti-avidin antibody (2.5 ~g/ml) was added, incubated for 40 minutes, washed, and subjected to a further 40-minute incubation with 100 ~I FrrC-avidin. After the last washings the slideswere mounted in phosphate-buffered saline covtainlng propidium iodide {1 ~g/ml) and examined immediately for fluorescent hybridization signals under U V light. 100-800 consecutive unselected cells were scored. RESULTS Patients The clinical, laboratory, and conventional cytogenetic findings of the patients are stlmmAT'iZed in Table 1. Patient 1 had trisomy 12 on cytogenetic analysisand was used as the pos itive control in all experiments.
0 and 1 hybridization signals on five separate experiments comprising a ~ of 800 cells were 1% and 7%, respectively. For the patient materials, these frequencies were somewhat higher, at 4.5% and 9.3%, respectively, probably reflecting deteriorationduring storage.Three hybridizationsi~rmlRwere found in 0.5% of cells in the control, 0.3-1.5% of cells in 10 patients,and none in seven patients.These patientswere considered to be negative. Patient 1 showed trisomy 12 in 12% of nmtephases o nconventional cyt~enetic RnA!~is, and in 26% of cells on ISH. Patient 8 showed normal metaphases on cytogenetic AnRIysis, but 9.6% of cells with three un ~tuivocal hybridization signals on four separate FISH experiments comprising 500 cells. This patient was considered to have a clone of cells with trisomy 12. DISCUSSION T h e relative rarity of CLL at 7% o f all l e u k e m i a i n o u r C h i o
In Situ Hybridlufion The results of in situ hybridization are summarized in Table 1. I n the normal control, the mean frequencies of finding
nase population accounted for the small number of cases we could investigate. Despite this, our study still illustrates several interesting points. First,in situ hybridization (FISH) has a high specificity
Trisomy 12 in CLL
for detection of trisomy 12, the false positive rate {three signals} being less than 1% of analyzed cells in the control and 2% in the patient materials. H S H successfully identified one case with trisomy 12 on conventional cytogenetic analysis, and also one case with normal conventional cytogenetic findings. In previous studies of CLL with normal conventional cytogenetics but trisomy 12 on FISH, 5-59% of interphase cells showed three hybridization signals [10-13]. This variation was also seen in our study, which might be due to the different types of preparations used. In our patients, we had no access to uncultured cells or cells from short-term (24hour) cultures. It is thus conceivable that in patient 8 the 3-day culture conditions were not optimal, so that only 10% of CLL cells survived, accounting for the normal cytogenetics (probably reflecting dividing T cells) and the rather low percentage of trisomy 12 cells. We had included cases with abnormal cytogenetics in our analysis, as Anastasi et al. [11] had shown that trisomy 12 clones might coexist with clones bearing other karyotypic aberrations. However, this phenomenon was not found in our patients. The use of ISH, w h e n compared with conventional cytogenotic analysis, increased our diagnostic yield of trisomy 12 from 5% to 10%, a twofold increase comparable to results in other series [10-12]. Although the number of patients in this study was small, the overall incidence of 10% of trisomy 12 is low compared to that of around 30% in the West. Further studies of oriental CLL patients will be required to confirm whether this low incidence of trisomy 12 in our patients reflects one of the biological differences of this rare disorder in the East as compared to the West. The authors thank A. Y. Y. Chan end T. S. K. Wen for technical assistence in conventional cytogenetic analysis. REFERENCES 1. Weias NS {1978):Geographical varietion in the incidence of the leukemias end lymphomas. In: Second Symposium on Epidemielegy and Cancer Registries in the Pacific Basin, Henderson BE, ed: Natl Cancer Inst Monogr 53. N.I.H. Publication N. 79-1864. 2. Wells R, Lau KS {1960):Incidence of leukemia in Singapore and rarity of chronic lymphocytic leukemia in Chinese. Br Med J 1:759. 3. Rai KR, Sawitsky A {1985):Diagnosis and treatment of chronic lymphocytic leukemia. In: Neoplastic Diseases of the Blood.
85
4.
5. 6. 7.
8. 9.
10.
Wiernik PH, Canellos GP, Kyle RA, Schiffer CA, eds. Churchill Livingstone, New York, pp. 105-120. Hen T, Ozer H, Sadamori N, Emrich L, Gomez GA, Henderson ES, Bloom ML, Sandberg AA (1984): Prognostic importance of cytngenetic abnormalities in patients with chronic lymphocytic leukemia. N Engl J Med 310:288-292. Bird ML, Uoshima Y, Rowley JD, Haren JM, ~miiman JW (1989): Chromosome abnormalities in B cell chronic lymphocytic leukemia and their clinical correlations. Leukemia 3:182-191. Geislar CH, Philip P, Hansen MM (1989): B-cell chronic lymphocytic leukemia: Clonsl chromosome abnormalities and prognosis in 89 cases. Eur J Haematol 1989;43:397-403. Juliusson G, Oscier DG, Fitchett M, Ross FM, Stockdill G, Mackie IvfJ, Parker AC, Castoldi GL, Cuneo A, Knuutila S, Elonan E, Gharton G (1990): Prognostic subgroups in B-cell chronic lymphocytic leukemia defined by specific chromosomal abnormalities. N Engl J Med 323:720-724. Crossen PE (1989): Cytogenetic and molecular changes in chronic B-cell leukemia. Cancer Genet Cytogenet 43:143-150. Sadamori N, Matsui S, Hen T, Sandbarg AA (1984): Comparative results with various polyclonal B-cell activators in aneuploid chronic lymphocytic leukemia. Cancer Genet Cytogenet 11:25-29. Losada AP, Wassman M, Tiainen M, Hopman AHN, Willard HF, Sol~ F, Caballin MR, Woessnar S, Knuutila S (1991): Trisomy 12 in chronic lymphocytic leukemia: an interphase cytogenetic study. Blood 78:775-779.
11. Anastasi J,Le Beau M M , Wardiman JW, Fernald AA, Larson RA, Rowley JD {1992): Detection of trisomy 12 in chronic lymphocytic leukemia by fluorescence in situ hybridization to interphase cells:a simple and sensitivemethod. Blood 79:1796-1801. 12. Cuneo A, Wlodarska I, Aly MS, Piva N, Carli MG, Fagioli F, Tallarico A, Pazzi I, FerrariL, Cassiman JJ,Van Den Berghe H, Castoldi GL (1992}:Non-radioactive in situ hybridization forthe detection and monitoring of trisomy 12 in B-cell chronic lymphocytic leukaemia. Br J Haematol 81:192-196. 13. Qumsiyeh MB, Thampel SA (1992}: Interphase detection of trisomy 12 in B-cell chronic lymphocytic leukemia by fluorescence hybridization in situ. Leukemia 6:602-605. 14. International Workshop on Chronic Lymphocytic Leukemia (1989}: Chronic lymphocytic leukemia: Recommendations for diagnosis, staging, and response criteria. A n n Intern M e d 110:236-238. 15. ISCN {1991}: Guidelines Cancer Cytogenetics. Supplement to A n International System for H u m a n Cytogenetic Nomenclature. Mitelman F, ed. Kargar, Basel, 1991. 16. Price CM, Kanfer EJ, Colman SM, Wastwood N, Barrett AJ,
Greaves MF (1991): Simultaneous genotypic and immunophenotypic analysis of interphase cells using dual-color fluorescence: a demonstration of lineage involvement in polycythemia vera. Blood 80:1033-1038.