No Evidence of Trisomy 12 or t(11;14) by Molecular Genetic Techniques in Chronic Lymphocytic Leukemia Cells with a Normal Karyotype Stefan Einhorn, Timothy Meeker, Gunnar Juliusson, Karin Burvall, and G0sta Gahrton
ABSTRACT: Chromosome analysis of B-cell mitogen-activated cells from patients with B-cell chronic lymphocytic leukemia (CLL) show clonal abnormalities in approximately one half of the cases. Among the most frequent aberrations are trisomy 12 and t(11;14). In the other half, no clonal chromosomal abnormalities are found. We wished to determine whether these CLL clones ore cytogenetically normal or whether the apparently normal karyotype results from an inability of the malignant ceils to enter metaphase in vitro, leaving only the few remaining normal cells to be karyotypically studied. Probes that can detect restriction fragment length polymorphisms (RFLPs) on chromosome 12, which makes it possible to determine the copy number of this chromosome, as well as bcl-1 probes that can detect t(11;14) were used. Of 13 CLL cases with normal karyotypes, none showed evidence for either trisomy 12 or t(11;14). This finding indicates that CLL cases with an apparently normal karyotype do not have two of the most common clonal chromosomal aberrations and may be karyotypically normal. The study also shows that Southern blot analyses, using probes that can detect BFLPs on chromosome 12, can be used for rapid and simple detection of trisomy 12 in CLL patients.
INTRODUCTION By using a panel of B-cell mitogens, metaphases in cells from most patients with B-cell chronic l y m p h o c y ti c leukemia can be induced (CLL) [1], and it has thus become possible to study chromosomal aberrations in this disease. These studies have s h o w n that approximately one third of the patients with chromosomal aberrations exhibit trisomy 12, sometimes as the sole aberration and sometimes together with other c h r o m o s o m a l abnormalities. Another relatively c o m m o n chromosomal aberration is t(ll;14)(q13;q32), observed in approximately 5% of CLL cases with clonal chromosomal abnormalities [1]. Other recurrent chromosomal changes include those involving c h r o m o s o m e s 6, 11, 13, and 14 [1]. In almost 50% of CLL cases, however, either no metaphase cells are observed or,
From Radiumhemmet, KarolinskaHospital, Stockholm,Sweden (S. E., K. B.),the Department of Medicine, University of California, San Francisco, California (T. M.), and the Department of Medicine, Huddinge, Hospital, Huddinge, Sweden (G. J., G. G.).
Address reprint requests to: Dr. Stefan Einhorn, Radiumhemmet, Karolinska Hospital, S-104 01 Stockholm, Sweden. Received June 27, 1989; accepted December 1, 1989. 183 © 1990 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet 48:183-192 (1990) 0165-4608/90/$03.50
184
s. E i n h o r n et al.
m o r e c o m m o n l y , c l o n a l c h r o m o s o m a l a b e r r a t i o n s are n o t o b s e r v e d i n t h e m e t a p h a s e s [1]. T w o p o s s i b i l i t i e s e x i s t to e x p l a i n t h e s e a p p a r e n t l y n o r m a l k a r y o t y p e s ; e i t h e r t h e c h r o m o s o m e s of t h e m a l i g n a n t c e l l s are c y t o g e n e t i c a l l y n o r m a l , or t h e B-cell m i t o g e n s h a v e n o t s u c c e e d e d i n p u s h i n g t h e CLL cells i n t o m e t a p h a s e , l e a v i n g o n l y t h e f e w r e m a i n i n g n o r m a l c e l l s to b e s t u d i e d k a r y o t y p i c a l l y . By u s i n g t w o bcl-1 p r o b e s t h a t c a n d e t e c t t(11;14} a n d t h r e e p r o b e s t h a t c a n d e t e c t r e s t r i c t i o n f r a g m e n t l e n g t h p o l y m o r p h i s m s (RFLPs) o n c h r o m o s o m e 12, m a k i n g it p o s s i b l e to d e t e r m i n e t h e c o p y n u m b e r of t h i s c h r o m o s o m e , w e i n v e s t i g a t e d t h e s e t w o p o s s i b i l i t i e s .
MATERIALS AND METHODS Patients T w e n t y - t w o p a t i e n t s w i t h B-cell CLL w e r e i n v e s t i g a t e d . C l i n i c a l a n d k a r y o t y p e d a t a are s h o w n i n T a b l e 1. S t a g i n g w a s p e r f o r m e d a c c o r d i n g to t h e m e t h o d of Rai et al. [2]. T w o p a t i e n t s h a d t r i s o m y 12, t w o h a d t(11;14), t w o h a d o t h e r c h r o m o s o m a l
Table 1
C l i n i c a l a n d k a r y o t y p e d a t a o n t h e CLL p a t i e n t s
Patient
Type
Phenotype
Stage
Leukocytes (x 10-9/L}
Previous treatment
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
CLL CLL CLL CLL CLL CLL CLL CLL CLL CLL CLL CLL CLL CLL CLL CLL PLL
~x ~8K 8K Smlg 8r /.L8K ~x ~8~ 8X ~SX ~.SK ~Sx ~x 8r ~ ~Sx /~SK
0 II I 0 I II 0 II II Ill 0 0 I II II II III
30 250 100 30 22 120 18 130 350 80 18 24 22 340 250 125 140
No Yes Yes No No Yes No Yes No Yes No No No No No No Yes
18
CLL/PLL
~8K
0
60
No
19
CLL
8X
IV
50
No
20 21
CLL CLL
i.~8x ~.K
I 0
18 30
No No
22
CLL
/z8r
0
60
No
Karyotype 46,XY 46,XY 46,XX 46,XY 46,XX 46,XX 46,XY 46,XY 46,XY 46,XX 46,XX 46,XY 46,XY ? ? v 46,XY, t(1;5)(q21;p15), t(6;6)(q23;q27), t(11;14}(q13;q32} 47,XX, + 3, del(17) (p12)t(11;14) (q13;q32) 47,XX, + 12, del(14)(q22) 46,X, - X, + 12 48,XY, + 3, + 18, del(21)(q21) 46,XX,t(13;13) (q31;q34)
Abbreviations: CLL, chronic lymphocytic leukemia; PLL, prolymphocytic leukemia.
QEvaluab|e metaphases compiled from the cultures that in the individual case were most successful.
No. of metaphases u 15 23 46 18 17 53 14 55 15 22 32 16 54 0 0 0 11
40
62 85 17 41
No Trisomy 12 or t(11;14) in CLL Cells with Normal Karyotype
185
aberrations, 13 had an apparently normal karyotype, and three showed no metaphase cells after stimulation by B-cell mitogens. Cell Separation Preparations of malignant cells were obtained from the peripheral blood by separation on Lymphoprep, followed by T-cell depletion by either E rosette sedimentation [3] or by separation on nylon wool [4]. After these procedures, the proportion of B-cells was always greater than or equal to 97%. Nonmalignant cells were obtained by three different procedures after Lymphoprep separation. First, T cells were enriched by E rosette sedimentation performed twice. The purity of these T-cell preparations was always greater than or equal to 80%, but the yield was low (1-5 million cells) in all cases except one. The contamination with B-cells was 5-20%. Second, granulocytes were separated by mixing the granulocyte/erythrocyte layer with 1 ml plasma and 0.4 ml dextran (Pharmacia, Uppsala, Sweden) 4.5% in saline. The mixture was kept at 4°C for 1-2 hours, after which granulocytes were recovered from the plasma fraction. The purity of these preparations was always more than 75%, but the yield was low (
ABSTRACT: Chromosome analysis of B-cell mitogen-activated cells from patients with B-cell chronic lymphocytic leukemia (CLL) show clonal abnormalities in approximately one half of the cases. Among the most frequent aberrations are trisomy 12 and t(11;14). In the other half, no clonal chromosomal abnormalities are found. We wished to determine whether these CLL clones ore cytogenetically normal or whether the apparently normal karyotype results from an inability of the malignant ceils to enter metaphase in vitro, leaving only the few remaining normal cells to be karyotypically studied. Probes that can detect restriction fragment length polymorphisms (RFLPs) on chromosome 12, which makes it possible to determine the copy number of this chromosome, as well as bcl-1 probes that can detect t(11;14) were used. Of 13 CLL cases with normal karyotypes, none showed evidence for either trisomy 12 or t(11;14). This finding indicates that CLL cases with an apparently normal karyotype do not have two of the most common clonal chromosomal aberrations and may be karyotypically normal. The study also shows that Southern blot analyses, using probes that can detect BFLPs on chromosome 12, can be used for rapid and simple detection of trisomy 12 in CLL patients.
INTRODUCTION By using a panel of B-cell mitogens, metaphases in cells from most patients with B-cell chronic l y m p h o c y ti c leukemia can be induced (CLL) [1], and it has thus become possible to study chromosomal aberrations in this disease. These studies have s h o w n that approximately one third of the patients with chromosomal aberrations exhibit trisomy 12, sometimes as the sole aberration and sometimes together with other c h r o m o s o m a l abnormalities. Another relatively c o m m o n chromosomal aberration is t(ll;14)(q13;q32), observed in approximately 5% of CLL cases with clonal chromosomal abnormalities [1]. Other recurrent chromosomal changes include those involving c h r o m o s o m e s 6, 11, 13, and 14 [1]. In almost 50% of CLL cases, however, either no metaphase cells are observed or,
From Radiumhemmet, KarolinskaHospital, Stockholm,Sweden (S. E., K. B.),the Department of Medicine, University of California, San Francisco, California (T. M.), and the Department of Medicine, Huddinge, Hospital, Huddinge, Sweden (G. J., G. G.).
Address reprint requests to: Dr. Stefan Einhorn, Radiumhemmet, Karolinska Hospital, S-104 01 Stockholm, Sweden. Received June 27, 1989; accepted December 1, 1989. 183 © 1990 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet 48:183-192 (1990) 0165-4608/90/$03.50
184
s. E i n h o r n et al.
m o r e c o m m o n l y , c l o n a l c h r o m o s o m a l a b e r r a t i o n s are n o t o b s e r v e d i n t h e m e t a p h a s e s [1]. T w o p o s s i b i l i t i e s e x i s t to e x p l a i n t h e s e a p p a r e n t l y n o r m a l k a r y o t y p e s ; e i t h e r t h e c h r o m o s o m e s of t h e m a l i g n a n t c e l l s are c y t o g e n e t i c a l l y n o r m a l , or t h e B-cell m i t o g e n s h a v e n o t s u c c e e d e d i n p u s h i n g t h e CLL cells i n t o m e t a p h a s e , l e a v i n g o n l y t h e f e w r e m a i n i n g n o r m a l c e l l s to b e s t u d i e d k a r y o t y p i c a l l y . By u s i n g t w o bcl-1 p r o b e s t h a t c a n d e t e c t t(11;14} a n d t h r e e p r o b e s t h a t c a n d e t e c t r e s t r i c t i o n f r a g m e n t l e n g t h p o l y m o r p h i s m s (RFLPs) o n c h r o m o s o m e 12, m a k i n g it p o s s i b l e to d e t e r m i n e t h e c o p y n u m b e r of t h i s c h r o m o s o m e , w e i n v e s t i g a t e d t h e s e t w o p o s s i b i l i t i e s .
MATERIALS AND METHODS Patients T w e n t y - t w o p a t i e n t s w i t h B-cell CLL w e r e i n v e s t i g a t e d . C l i n i c a l a n d k a r y o t y p e d a t a are s h o w n i n T a b l e 1. S t a g i n g w a s p e r f o r m e d a c c o r d i n g to t h e m e t h o d of Rai et al. [2]. T w o p a t i e n t s h a d t r i s o m y 12, t w o h a d t(11;14), t w o h a d o t h e r c h r o m o s o m a l
Table 1
C l i n i c a l a n d k a r y o t y p e d a t a o n t h e CLL p a t i e n t s
Patient
Type
Phenotype
Stage
Leukocytes (x 10-9/L}
Previous treatment
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
CLL CLL CLL CLL CLL CLL CLL CLL CLL CLL CLL CLL CLL CLL CLL CLL PLL
~x ~8K 8K Smlg 8r /.L8K ~x ~8~ 8X ~SX ~.SK ~Sx ~x 8r ~ ~Sx /~SK
0 II I 0 I II 0 II II Ill 0 0 I II II II III
30 250 100 30 22 120 18 130 350 80 18 24 22 340 250 125 140
No Yes Yes No No Yes No Yes No Yes No No No No No No Yes
18
CLL/PLL
~8K
0
60
No
19
CLL
8X
IV
50
No
20 21
CLL CLL
i.~8x ~.K
I 0
18 30
No No
22
CLL
/z8r
0
60
No
Karyotype 46,XY 46,XY 46,XX 46,XY 46,XX 46,XX 46,XY 46,XY 46,XY 46,XX 46,XX 46,XY 46,XY ? ? v 46,XY, t(1;5)(q21;p15), t(6;6)(q23;q27), t(11;14}(q13;q32} 47,XX, + 3, del(17) (p12)t(11;14) (q13;q32) 47,XX, + 12, del(14)(q22) 46,X, - X, + 12 48,XY, + 3, + 18, del(21)(q21) 46,XX,t(13;13) (q31;q34)
Abbreviations: CLL, chronic lymphocytic leukemia; PLL, prolymphocytic leukemia.
QEvaluab|e metaphases compiled from the cultures that in the individual case were most successful.
No. of metaphases u 15 23 46 18 17 53 14 55 15 22 32 16 54 0 0 0 11
40
62 85 17 41
No Trisomy 12 or t(11;14) in CLL Cells with Normal Karyotype
185
aberrations, 13 had an apparently normal karyotype, and three showed no metaphase cells after stimulation by B-cell mitogens. Cell Separation Preparations of malignant cells were obtained from the peripheral blood by separation on Lymphoprep, followed by T-cell depletion by either E rosette sedimentation [3] or by separation on nylon wool [4]. After these procedures, the proportion of B-cells was always greater than or equal to 97%. Nonmalignant cells were obtained by three different procedures after Lymphoprep separation. First, T cells were enriched by E rosette sedimentation performed twice. The purity of these T-cell preparations was always greater than or equal to 80%, but the yield was low (1-5 million cells) in all cases except one. The contamination with B-cells was 5-20%. Second, granulocytes were separated by mixing the granulocyte/erythrocyte layer with 1 ml plasma and 0.4 ml dextran (Pharmacia, Uppsala, Sweden) 4.5% in saline. The mixture was kept at 4°C for 1-2 hours, after which granulocytes were recovered from the plasma fraction. The purity of these preparations was always more than 75%, but the yield was low (
