Cytogenetic Analysis of a United Kingdom Series of Non-Hodgkins Lymphomas David W. Hammond, John R. Goepel, Muriel Aitken, Barry W. Hancock, Anthony M. Potter, and Malcolm H. Goyns
ABSTRACT: We describe cytogenetic analyses of cells derived from 40 non-Hodgkins lymphoma (NHL)
node biopsies, 23 of which were from patients who had not been treated before biopsy. We noted that the chromosomes most frequently gained were X (32%), 12 (27%), and 3 (24%). Monosomies were much less common; loss of chromosome 13 (13.5%) was most frequent. Structural abnormalities primarily involved chromosomes 14 (70%), 1 (40.5%), 18 (38%), 6 (35%), and 17 (22%). Low- and high-grade disease showed similar patterns of structural changes; however, a markedly greater number of chromosome gains were associated with low-grade disease. Biopsy samples from patients who had previously been treated showed an increased frequency of structural abnormalities, as well as a significantly larger n u m b e r of chromosome gains. The importance of these observations, particularly with regard to possible oncogene involvement in lymphoma evolution, is discussed.
INTRODUCTION
MATERIALS A N D METHODS
Cytogenetic analyses of tumor cells have previously indicated that in m a n y cases a close relationship exists between specific c h r o m o s o m a l abnormalities and particular types of malignancy. This has become even more relevant in recent years w i t h the correlation of m a n y of these abnormalities to activation or deletion of oncogenes. Although some c h r o m o s o m a l changes occur very frequently, m a n y others occur more sporadically. In an attempt to determine the true significance of the latter, m a n y samples must be analyzed. In non-Hodgkins l y m p h o m a (NHL), several such studies have been p u b l i s h e d from centers in the United States [1-4], Canada [5], S w e d e n [6], France [7], the U.S.S.R. [8], Australia [9], and Japan [10]. Studies have also analyzed data gathered from several countries [11]. These investigations have been able to identify a number of nonr a n d o m abnormalities but have also raised more questions, not least that regarding regional variation in karyotypes [11, 12]. We report 40 karyotypes of cells isolated from NHL biopsies, a series we believe to be the largest yet reported from the United Kingdom.
Patients
From the Department of Clinical Oncology, Royal Hallamshire Hospital, (D. W. H., M. A., B. W. H., M. H. G.), the Centre for Human Genetics, (D. W. H., A. M. P.), and the Department of Pathology, University Medical School, Sheffield (J. R. G.), England. Address reprint requests to: Dr. Malcolm H. Goyns, Department of Clinical Oncology, Royal Hallamshire Hospital, Sheffield, $10 2JF, England. Received November I, 1991; accepted January 22, 1992. © 1992 Elsevier Science Publishing Co., Inc.
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Between 1989 and 1991, a consecutive series of 40 node biopsies, obtained from 37 white NHL patients, were studied cytogenetically. Twenty-three of the patients in this study were not treated before biopsy, but 17 of the s a m p l e s represent relapse cases who had been diagnosed p r e v i o u s l y and who had undergone some form of treatment. The diagnostic classification system used in this s t u d y was the updated Kiel classification [13]. Clinical details for each of the patients, relating to staging of the disease at presentation, types of treatment before biopsy, and diagnosis are s u m m a r i z e d in Table 1. Cytogenetic Methods
L y m p h node biopsy material was collected in Hams F10 medium, buffered with 25 mM HEPES. The tissue s a m p l e was cut with scissors to obtain cell suspensions. The latter were used to set up three cultures in p r e w a r m e d Hams F10 m e d i u m s u p p l e m e n t e d with 10% (vol/vol) p o o l e d h u m a n serum. Culture 1 had 0.1 ~g/ml Colcemid a d d e d before being incubated at 37°C for 30 minutes. Culture 2 had 0.01 ~g/ml Colcemid a d d e d before being incubated at 37°C overnight. Culture 3 was incubated overnight at 37°C and then had 0.1 /zg/ml Colcemid added, before being incubated for another hour. In all cases, the cells were centrifuged, r e s u s p e n d e d in 0,075 M KCI for 10 m i n u t e s at 37°C, and then centrifuged again before being fixed in 3 : 1 methan o l : a c e t i c acid. The cells were d r o p p e d onto glass slides (previously cleaned in Decon and stored in distilled water 31 Cancer Genet Cytogenet 61:31 38 (1992) 0165-46o8/92/$05.0o
32
Table 1
Patient
D . W . H a m m o n d et al.
Clinical details of patients Treatment Staging at before presentation biopsy
L001 L002 L003 L004 L005 L006 L007 L008 L009 L010A L010B L011 L012 L013 L014 L015 L016 L017 L018 L019 L020A L020B L021 L022 L023 L024 L025 L026A L026B L027 L028 L029 L030 L031 L032 L033 L034 L035 L036 L037
IIA IVA IVB IA IA [VA IA IIIA IA IIIB IA IVA IVA IVA IVA IVA IIIA IIIA IIA 1VA -IVB IIIA IIA ND IIIA IVA -IVA IVB IIIA IVB ND IIA IVB IVA IIIB IA IA
NT OC NT NT NT CC NT NT RT CC OCP OC OCP NT NT NT NT CC NT NT OC OCP NT OC NT NT RT NT NT NT NT OC NT NT OC NT OC CC OC NT
Diagnosis B-cell high-grade centroblastic B-cell low-grade follicular B-cell high-grade centroblastic B-cell low-grade follicular B-cell low-grade follicular B-cell high-grade centroblastic B-cell low-grade follicular B-cell low-grade follicular B-cell low-grade follicular B-cell low-grade follicular B-cell high-grade centroblastic B-cell low-grade centrocytic B-cell low-grade lymphocytic B-cell low-grade follicular B-cell low-grade follicular B-cell low-grade lymphocytic B-cell low-grade lymphocytic B-cell high-grade centroblastic B-cell low-grade lymphocytic T-cell low-grade T zone B-cell low-grade lymphocytic ~13-celllow-grade lymphocytic B-cell high-grade centroblastic B-cell low-grade follicular B-cell low-grade follicular B-cell high-grade immunoblastic B-cell high-grade centroblastic B-ceil low-grade lymphocytic B-cell low-grade lymphocytic B-cell low-grade follicular B-cell high-grade centroblastic B-cell high-grade centroblastic B-cell high-grade centroblastic B-cell low-grade follicular B-cell low-grade follicular T-cell high-grade immunoblastic B-cell low-grade follicular B-cell high-grade immunoblastic B-ceil low-grade follicular B-cell low-grade follicular
Abbreviations: CC, intravenous administration of combination chemother-
apy; ND, not determined; NT, no treatment; OC, oral administration of chlorambucil; OCP. oral administration of chlorambucil and prednisolone; RT, radiotherapy.
at 4°C) blotted, air-dried, and aged at 80°C. The chromosomes were G-banded with trypsin and Leishman stained [14]. Forty-one node samples from 38 patients were investigated in this way, of w h i c h 40 yielded analysable metaphases, a success rate of almost 100%. The karyotypes were described according to the International System for H u m a n Cytogenetic N o m e n c l a t u r e (ISCN) [15].
RESULTS The karyotypes of cells isolated from each of the node biopsies, the n u m b e r of cells e x a m i n e d and the proportion of normal cells observed are s u m m a r i z e d in Table 2. Only three of the specimens analyzed exhibited exclusively normal karyotypes. The r e m a in d e r contained cells with a variety of numerical and/or structural abnormalities. In about
half of the samples, a m i x of normal and abnormal cells was observed. Figure 1 shows the percentage of cases in w h i c h a gain or loss of each of the c h r o m o s o m e s was observed. The most frequently gained c h r o m o s o m e was X (32%), w h i c h was observed in both males (25%) and females (33%). This was followed in frequency by trisomy of c h r o m o s o m e s 12 (27%), 3 (24%), 7 (16%), and 8 (16%). M o n o s o m y was far less c o m m o n , with the most frequent loss i n v o l v i n g chrom o s o m e 13 (13.5%). With the e x c e p t i o n of L028 and L032, w h i c h both exhibited very c o m p l e x karyotypes w i t h several marker c h r o m o s o m e s , loss of a c h r o m o s o m e 11, 13, or 17 appeared to be m u t u a l l y exclusive. As a result, a quarter of the samples studied s h o w e d a loss of one of these chromosomes. Figure 2 summarizes the percentage of cases in w h i c h structural abnormalities were observed. With the exception of c h r o m o s o m e s 21 and Y, structural abnormalities inv o l v e d all chromosomes. The most frequently affected c h r o m o s o m e was 14 (70%), w h i c h in a p p r o x i m a t e l y half of these samples was i n v o l v e d in the t(14;18)(q32;q21). This correlated exactly with the presence of the reciprocal derivative c h r o m o s o m e s 1 8 q - . In some cases (11%), however, an apparently typical 1 4 q + derivative c h r o m o s o m e was observed, but the reciprocal 1 8 q - was absent. Several abnormalities were derived from c h r o m o s o m e 1 (40.5%), w h i c h in many cases appeared to i n v o l v e bands p 3 2 - 3 6 or q21. A b n o r m a l c h r o m o s o m e s 6 were observed in 35% of cases and most exhibited an altered q arm, often as a result of a deletion. Other c o m m o n l y rearranged c h r o m o s o m e s were 9 (22%), 17 (22%), and 3 (19%). In three cases (LOIOB, L020B, L026B), we were able to obtain second biopsy samples for analysis. The data from these were not i n c l u d e d in the analyses s h o w n in Figs. 1-4. Such data are of interest because of the possible insight they may provide into the e v o l u t i o n of the disease. In two cases (L020 and L026) there was little change in the disease or in the respective karyotypes. In the third case, however, the disease had transformed from low-grade (LO10A) to high-grade (LO10B), and this was paralleled by very significant changes in the karyotype. This series contained both high- and low-grade l ymphomas (Table 1), allowing c o m p a r i s o n b e t w e e n the karyotypes of these two classes of NHL. Because cases of loss of ch r o m o so m es were relatively few they have not been considered; however, n u m e r i c a l gains are s u m m a r i z e d in Fig. 3, w h i c h shows a remarkable difference b et w e e n lowand high-grade NHL, for most of c h r o m o s o m e gains appeared to be concentrated in the low-grade samples, although additional c h r o m o s o m e s 3 and X occurred at similar frequencies in both groups. Comparison of structural abnormalities s h o w e d a similar pattern in both groups, with only a slightly higher frequency in the low-grade samples. The series could also be split into a p p r o x i m a t e l y two equal-sized groups on the basis of treatment before biopsy. Again there were relatively few c h r o m o s o m e losses to analyze, but n u m er i cal gains are s u m m a r i z e d in Fig. 4. The samples from patients w h o had previously been treated, exhibited an increased frequency of gains of all chromo-
33
Table 2
Karyotypes of cells from NHL node biopsies
Patient
No. of cells examined
Percentage normal
L001
25
0
LO02 L003
29 34
0 0
LO04
49
L005
40
27
L006 L007
22 28
18
L008
18
0
LO09
24
12
L010A
25
0
L010B
18
0
L011
28
18
L012
28
18
L013 L014
22 22
0 18
L015 L016
23 24
0 4
L017
31
7
L018 L019
32 35
100 6
L020A
27
4
LO20B L021
17 27
0 15
L022
24
0
25
0
Karyotype
47,XY, + 3,dup(1)(qllq44),del(2){q21),del(9)(q21}, der(11}t(11;?)(q23;?) 46,XX,inv(9)(p13q22},t(14;18)(q32;q21),der(16)t(16;?)(p13.3;?) 46,XY, + X,der(3)t(3;?)(q13;?),der(6)t(6;?)(q13;?), - 8, del(14)(q22q24),del(17)(p13) 46,XY 46,XY,del(5)(p14),der(12)t(12;?)(p11;?),der(15)t(15;?)(q22;?} 46,XX 48,XX,+ 8,+ 12 4 9 , X X , + X , + 8 , + 12 46,XX,del(14)(q31),t(20;22)(q13.3;q12),der(14)t(14;?)(q32;?) 46,XY 94,XXYY, + X x 2,der(1)t(1;?)(p13;?),der(2)t(2;?)(p21;?) x 2,der(3)t(3;?) (q13;?), - 4,del(6)(q16q22), + 12 x 2, - 17,t(14;18)(q32;q21) x 2 48,XX, + 3,del(6)(p21), + del(6)(q21),t(10;13)(q22;q12),del(14)(q31), del(17)(p11),der(22)t(22;?)(q13;?) 46,XX 62,XX, + der(1)t(1;?)(p11 ;?), + 2, + ins(3;?)(q21;?) x 2, + del(5)(q31), + del(7)(q32) x 2, + 8, + 9,del(10)(q22),der(10)t(10;?)(q23;?), + 11, + 12, + 13,t(14;18)(q32;q21), + der(14)t(14;?)(q32;?), + 19, + 20, + 21 92,XXXX, + X,der(1}t(1;?)(p36;?), + del(3)(p21}, der(3)t(3;?)(p13;?),del(6)(q15q21),t[del(9)(p22);13](q21;q12), dup(12)(q13q22),der(13)t[del(9)(p22);13](q21;q12), t(14;18)(q32;q21),- 1 5 , - 16,der(16}t(16;?)(q12;?),der(19}t(19;?)(p11;?) 48,XX, + der(X)t(X;?)(p22;?),der(1)t(1;?)(p36;?),der(3},t(3;?)(q21;?), del(6)(q15q21), + 7,dup(12)(q13q22),der(13)t[del(9)(p22);13] (q21;q12),t(14;18)(q32;q21),der(19)t(19;?)(q13.4;?) 46,XY 49,XY, + 8, + 9,der(14)t(14;?)(q32;?), + 18 49,XY,ins(1)(q21q257), + 8, + 9,der(14)t(14;?)(q32;?), + 18 49,XY, + 8, + i(9q),der(14)t(14;?)(q32;?), + 18 46,XY 48,XY,der(1)t(1;?)(p32;?), + 7, - 11,del(14)(q24),del(15)(q24), del(20(q13), + 2mar 4 5 , X , - X , + 1 2 , - 14 46,XX 46,XX,t(6;8)(p21;q24},t(14;18)(q32;q21) 47,XX,t(6;8)(p21;q24), + 8,t(14;18)(q32;q21) 48,XX,t(14;18)(q32;q21), + 18, + der(18)t(18;?)(q21;?) 46,XX 47,XX, + 12,del(14)(q22} 46,XY 47,XY, + X,der(6)t(6;?)(q23;?),ins(7;?)(p13;?),t(9;17)(q11;p11), + 1 2 , - 13,t(14;18)(q32;q21),- 15, + 18 46,XY 46,XY 46,XY, - 5, - 9,der(10)t(10;?)(p11;?),der(11)t(11;?)(q21;?), der(16)t(1;16)(q21;p11), + 2mar. 46,XX 46,XX,der(11}t(ll;?)(q13;?) 46,XX,der(11)t(11;?)(q13;?) 46,XY 47,XY,der(1)t(1;?)(q21;?),der(5)t(5;?)(p13;?),- 8 , - 9 , - 10, - 1 1 , - 13,del(13q31),der(14)t(14;?)(q24;?), + der(17)t(17;?) (p12;?), + 22, + 4mar 50,XX, + X,der(1}t(1;9)(p32;q12),der(5)t(5;?)(q13;?), + der(5}t(5;?)(q13;?),der(6)t(6;?;5)(q13;?;q13), - 8, + 12,der(14)t(14;?)(q32;?) x 2,der(15)t(1;15)(q21;q22), + 18, + mar
(continued)
34
D.W. Hammond
Table 2
K a r y o t y p e s of cells f r o m N H L n o d e b i o p s i e s
Patient
No. of cells examined
Percentage normal
L023
29
14
L024 L025
20 23
0 22
L026A L026B L027
25 21 19
0 0 11
L028
29
14
L029
35
0
L030 L031
23 36
0 17
L032
32
50
L033 L034
59 24
97 13
L035 L036
37 33
100 9
L037
21
19
et al.
(Continued) Karyotype
46,XX 53-55,XX, + X,dup(1)(q21q32),der(1)t(1;?)(p32:?), der(2)t(2;?)(p21;?), + 3, + 5,i(6p), + 7, + der(8)t(8;?)(p11;?), + 11,t(13q15q),t(14;18)(q32;q21),- 15,der(18)t(18;?)(q21;?), + 21, + 2r 47,XY,der(1)t(1;?)(q42;?},del(2)(q23),t(8;14)(q24;q32), + 21 46,XX 51,XX,dup(1)(qllq25),der(2}t(2;?)(p23;?), + 3, + 3, der(22)t(22;?)(q13;?), + 3mar 46,XY, + 1 2 , - 13,der(17)t(17;?)(q21;?) x 2 46,XY, + 1 2 , - 13,der(17)t(17;?)(q21;?) x 2 46,XX 48,XX, + 5,i(6p), + 12,t(14;18)(q32;q21), - 18, + mar 46,XX 43-46,X,der(X)t(X;?)(p22;?),der(1)t(1;?)(p32;?), + 3,der(4)t(4;?)(q21;?), - 6,der(7)t(7;?)(p21 ;?), der(8)t(8;?)(p21;?),- 1 0 , - 13,der(16)t(16;?)(p12;?), 17,der(18)t(18;?)(q23;?),der(20)t(X;20;?)(q13,q13;?),- 22, + 3mar 88,XXYY, - 5,del(6)(q23q26), - 9, - 10, - 13,t(14;18)(q32;q21), - 16, del(16)(q22), - 20, + 2mar 49,XY, + X, + 12, + mar 46,XX 46,XX,der(14)t(14;?)(q32;?) 46,XX,der(3)t(3;?}(q27;?),der(14}t(14;?)(q32;?) 46,XX,der(14)t(14;?)(q32;?),del(17)(p13) 46,X,del(X)(q28),del(2)(p23),der(14)t(14;?)(q32;?),del(20)(p12) 46,XY 79-82,XXY,- Y,i(1q),der(1)(1;?)(p36;?) × 2,der(1)(1;?)(p36;?), der(2)t(2;?)(p13;?),i(3p), + der(3)t(3;?)(p13;?), - 4, del(4)(q12q25), + del(6)(q22q23) × 2,del(7)(q22) × 2, 8,der(8)t(8;?)(p11;?),- 9,der(9)t(9;?)(q34;?),- 11 x 2, - 12,der(12)t(12;?)(p13;?),- 13,der(14)t(14;?)(q22;?), der(16)t(16;?)(q11;?) × 2, + i n v ( 1 6 ) ( q l l p 1 2 ) , - 1 8 , - 1 9 , - 21, + 3mar 46,XX 46,XX 49,XX, +X, ÷ 7, + 8,der(6)t(6;?)(q23;?) x 2, t(14;18)(q32 ;q21),der(17)t(17 ;?)(q21 ;?) 46,XY 46,XX 74,XX, + X × 2, + der(1)t(1;?)(p13;?), + 2, + der(3)t(3;?)(q27;?), + 4, + 5, + 6, + der(6)t(6;?)(q23;?), + 7, + 8, + 9, + 11, + 13, + 16, t(14;18)(q32;q21) × 2 , - 19, + 20, + del(22)(q11), + 11mar 46,XX 46,XX,t(14;18)(q32;q21) 46,XX,t(14;18)(q32;q21),t(9;19)(p13;q13.1)
s o m e s , w i t h t h e e x c e p t i o n of c h r o m o s o m e 12 a n d , to a m u c h l e s s e r e x t e n t , c h r o m o s o m e s 21 a n d 22. O c c u r r e n c e of structural changes showed a similar pattern in both groups, a l t h o u g h t h e y a p p e a r e d to b e m o r e c o m m o n i n t h e t r e a t e d group. Low-grade samples in particular showed more struct u r a l a b n o r m a l i t i e s after t r e a t m e n t t h a n t h e h i g h - g r a d e group.
D I S C U S S I O N
W e r e p o r t t h e c y t o g e n e t i c a n a l y s e s of a series of 40 NHL n o d e b i o p s i e s o b t a i n e d f r o m U.K. p a t i e n t s . T h i s g r o u p w a s c o m p o s e d of b o t h l o w - a n d h i g h - g r a d e N H L s a m p l e s , a n d
a p p r o x i m a t e l y e q u a l n u m b e r s of n o n t r e a t e d a n d p r e v i o u s l y treated patients. The most striking finding was the much g r e a t e r p r e v a l e n c e of n u m e r i c a l g a i n s i n t h e l o w - g r a d e N H L s a m p l e s . T w i c e as m a n y of t h e s e s a m p l e s e x h i b i t e d trisom i e s of s e v e r a l c h r o m o s o m e s t h a n d i d t h o s e i n t h e h i g h g r a d e g r o u p . F u r t h e r m o r e , t r i s o m i e s of a l m o s t all t h e c h r o mosomes were observed in low-grade samples, whereas o n l y s e v e n of t h e c h r o m o s o m e s w e r e i n v o l v e d i n t r i s o m i e s in the high-grade group. To our knowledge, this observation h a s n o t b e e n p r e v i o u s l y d o c u m e n t e d . It m a y b e a n i n d i c a t i o n of a m o r e g e n e r a l b r e a k d o w n i n t h e m a c h i n e r y of cell r e p l i c a t i o n i n l o w - g r a d e d i s e a s e or, as t h i s effect h a s n o t b e e n n o t e d before, it m a y r e p r e s e n t a n e x a m p l e of r e g i o n a l
NHL Karyotypes
35
35-: 30- ~1 20-
]5-
lo-
50
1 ' 2 ' 3 ' 4 ' 5 ' 6 ' 7 ' 8 ' 9 ' 1 0 ' 1 1 ' 1 2 ' 1 3 ' 1 4 ' 1 5 ' 1 6 ' 1 7 ' 1 8 ' 1 9 ' 2 0 ' 2 1 ' 2 2 ' X Y;
Figure 1 Analysis of numerical changes in NHL node biopsy samples. (a) Percentage of cases exhibiting chromosome gains; (b) percentage of cases exhibiting chromosome losses.
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1 2 3 4 5 6 7 8 9 10111213141516171819202122X Figure 2
Y
Percentage of cases exhibiting chromosomal structural changes in NHL node biopsy samples.
353025 20 15 10 50
a
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Figure 3 Comparison of numerical gains of chromosomes in high- and low-grade NHL. (a) Percentage of highgrade cases exhibiting chromosome gains; (b) percentage of low-grade cases exhibiting chromosome gains.
36
D . W . H a m m o n d et al.
35 3025 20 15 105 i
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2 . 3 . 4 . 5. 6. .7 .8 . 9 . 1 0. 1 1. 1 2. 1 3. 1 .4 1.5 1.6 1.7 1. 8 1. 9 2. 0 2 1
22' X Y
Figure 4 Comparison of numerical gains of chromosomes in biopsy samples from nontreated and treated NHL patients. (a) Percentage of nontreated cases exhibiting numerical gains; (b) percentage of treated cases exhibiting numerical gains.
variation [11, 12]. Further analysis of our data, however, s h o w e d that trisomies a p p e a r e d to be more c o m m o n in low-grade disease samples from patients who had been p r e v i o u s l y treated than in similarly treated high-grade sampies. The effect of treatment m a y therefore have had a m u c h greater effect on the c h r o m o s o m e s in low-grade than in high-grade NHL. The most c o m m o n n u m e r i c a l c h r o m o s o m e change observed was the presence of an a d d i t i o n a l c h r o m o s o m e X, present at a higher frequency than in previously reported series, in w h i c h the frequency varied between 3 and 22.5% [3, 4, 8]. The significance of an extra c h r o m o s o m e X is unclear, although studies of other types of malignancy have i n d i c a t e d that a d d i t i o n a l X chromosomes are active in neoplastic cells [16, 17]. In some NHL cases, an extra chromosome X has been reported to be the only identifiable abnorm a l i t y [18]. Trisomies involving c h r o m o s o m e s 3 and 12 were observed at frequencies similar to those previously reported [3, 4, 8, 11]. Our data do not support the suggestions that + 3 is associated with high-grade l y m p h o m a s [5, 8], or that + 12 is associated with l y m p h o c y t i c [18] or diffuse large cell l y m p h o m a [3, 19]. In our series, + 12 was twice as c o m m o n in low-grade NHL but was not particularly associated with the l y m p h o c y t i c subtype. Alone among the c h r o m o s o m e gains, + 12 was more frequent in patients w h o had not been treated; even though low- and high-grade samples were evenly distributed between the treated and nontreated groups. Loss of a whole c h r o m o s o m e d i d not appear to be a frequent occurrence; however, further analysis demonstrated that w h e n loss of c h r o m o s o m e s 11, 13, or 17 was observed, u s u a l l y only one of these c h r o m o s o m e s was lost in any particular sample. This is n o t e w o r t h y because m a n y studies of other malignancies have demonstrated the pres-
ence of t u m o r supressor genes on these chromosomes. Chromosome 13 carries the RB-1 gene [20, 21], c h r o m o s o m e 17 the p53 gene [22], and c h r o m o s o m e 11 the W i l m s ' tumor gene [23]. That loss of these c h r o m o s o m e s occurs in a quarter of the cases in our study suggests that further m o l e c u l a r biology studies of the i m p l i c a t e d t u m o r s u p p r e s s o r genes may be informative. In comparison, deletions of parts of c h r o m o s o m e s were more frequent. In particular, c h r o m o s o m e 6 was often observed to have part or all of its q arm deleted. If translocation breakpoints are also i n c l u d e d , 27% of all cases exhibited deletion or rearrangements of 6q21-25. Similar findings have been d o c u m e n t e d p r e v i o u s l y [3, 8, 11]. We found no evidence to s u p p o r t the suggestions that these abnormalities were p r i m a r i l y associated either w i t h p r e v i o u s treatment [19] or i m m u n o b l a s t i c l y m p h o m a [5]. Indeed, our data suggested a more c o m m o n association of 6q abnormalities in low-grade disease. Structural abnormalities of 6q have been observed in a w i d e range of m a l i g n a n t cells [18], and several protooncogenes have been localized to this region [24]. The most extensively s t u d i e d of these is MYB which, as a result of the 6 q - deletions, has been observed to be altered in a n u m b e r of l y m p h o m a cell lines [25]. Whether MYB is the target gene associated w i t h the 6q abnormalities is still uncertain, however. From the c h r o m o s o m a l abnormalities observed in this series, several other oncogenes c o u l d be i m p l i c a t e d in the evolution of NHL; e.g., we noted l p 3 2 - 3 6 (L-MYC), 17p13 (p53), and 19q13 (BCL-3) breakpoints in several cases. In only one case, however, d i d we observe a translocation involving 11q13 (BCL-1). The most c o m m o n c h r o m o s o m a l a b n o r m a l i t y we detected was the t(14;18)(q32;q21}, w i t h frequencies of 59% in follicular low-grade and 17% in high-grade NHL. These
NHL Karyotypes
figures are in agreement w i t h results of some previously p u b l i s h e d studies [3, 5], but lower than those of others [4, 8]. This translocation appears to be the most c o m m o n m e c h a n i s m by w h i c h the BCL-2 gene is activated [26]. Although the frequency of the translocation in our series was not as high as reported in some others [4, 8], we did note that in 11% of the cases an apparently typical 14q + derivative c h r o m o s o m e occured w i t h o u t a reciprocal 1 8 q - . The latter may have been lost or masked by further translocation events in these cells, w h i c h w o u l d mean that the frequency of the t(14;18) may actually be higher in our series. This situation highlights the problems of analyzing c o m p l e x karyotypes, such as those w h i c h occur in NHL, w h e n banding patterns alone are used. Further advances in understanding abnormal c h r o m o s o m e structure may therefore require application of n e w m e t h o d s such as nonradioactive in situ hybridization of DNA probes [27].
This work was supported by the Yorkshire Cancer Research Campaign.
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10. Konishi H, Sakurai M, Nakao H, Maseki N, Kaneko Y, Yagiri Y, Notohara K, Frizzera G (1990): Chromosome abnormalities in malignant lymphoma in patients from Kurashiki: Histological and immunophenotypic correlations. Cancer Res 50: 2698-2703. 11. Fifth International Workshop on Chromosomes in LeukaemiaLymphoma (1987): Correlation of chromosome abnormalities with histologic and immunologic characteristics in non-Hodgkins lymphoma and adult T-cell leukemia-lymphoma. Blood 69:97-102. 12. Johansson B, Mertens F, Mitelman F (1991): Geographic heterogeneity of neoplasia-associated chromosome aberrations. Genes Chrom Cancer 3:1-7. 13. Stansveld AG, Diebold J. Kapanci Y, Kelenyi G, Lennert K, Mioduszewska O, Noel H (1988): Updated Kiel classification for lymphomas. Lancet 1:292-293. 14. Seabright M (1971): A rapid banding technique for human chromosomes. Lancet 2:971-972. 15. ISCN (1985): An International System for Human Cytogenetic Nomenclature, Harnden DG, Klinger HP (eds); published in collaboration with Cytogenet Cell Genet (Karger, Basel, 1985); also in Birth Defects: Original Article Series, Vol. 21, No. 1 (March of Dimes Birth Defects Foundation, New York, 1985). 16. Dutrillaux H, Muleris M, Gerbault-Seureau M (1986): Imbalance of sex chromosomes, with gain of early-replicating X, in human solid tumours. Int J Cancer 38:475-479. 17. Wang N, Cedrone E, Skuse GR, Insel R, Dry J (1990): Two identical active X chromosomes in human mammary carcinoma cells. Cancer Genet Cytogenet 46:271-280. 18. Mitelman F, Kaneko Y, Trent JM (1990): HGM 10.5, Report of the committee on chromosomal changes in neoplasia. Cytogenet Cell Genet 55:358-386. 19. Cabanillas F, Pathak S, Manning J, Katz R, McLaughlin P, Velasquez WS, Hagemeister FB, Goodacre A, Cork A, Butler JJ, Freireich EJ (1988): Frequent non random chromosome abnormalities in 27 patients with untreated large cell lymphoma and immunoblastic lymphoma. Cancer Res 48:55575564. 20. Lundberg C, Skoog L, Cavanee WK, Nordenskjold M (1987): Loss of heterozygosity in human ductal breast tumors indicates a recessive mutation on chromosome 13. Proc Natl Acad Sci USA 84:2372-2376. 21. Bookstein R, Lee EYHP, To H, Young LJ, Sery TW, Hayes RC, Friedmann T, Lee WH (1988): Human retinoblastoma susceptibility gene: Genomic organisation and analysis of heterozygous intragenic deletion mutants. Proc Natl Acad Sci USA 85:2210-2214. 22. Baker SJ, Fearon ER, Nigro JM, Hamilton SR, Preisinger AC, Jessup JM, van Tuinen P, Ledbetter DH, Barker DF, Nakamura Y, White R, Vogelstein B (1989): Chromosome 17 deletion and p53 gene mutation in colorectal carcinomas. Science 244: 217-221. 23. van Heyningen V, Boyd PA, Seawright A, Fletcher JM, Fantes JA, Buckton KE, Spowart G, Porteus DJ, Hill RE, Newton RS, Hastie MD (1985): Molecular analysis of chromosome 11 deletions in aniridia-Wilms tumor syndrome. Proc Natl Acad Sci USA 82:8592-8596. 24. Spence MA, Spurr NK, Field LL (1989): HGM 10; Report of the committee on the genetic constitution of chromosome 6. Cytogenet Cell Genet 51:149-165. 25. Barletta C, Pelicci PG, Kenyon LC, Smith SD, Dalla-Favera R (1987): Relationship between the c-myb locus and the 6 q chromosomal aberration in leukemias and lymphomas. Science 235:1064-1067. 26. Tsujimoto Y, Gorham J, Cossman J, Jaffe E, Croce CM (1985):
38 Involvement of the bcl-2 gene in human follicular lymphoma. Science 228:1440-1443. 27. Smit VTHBM, Wessels JW, Mollevanger P, Dauwerse JG, Vliet M, Beverstock GC, Breuning MH, Devilee P, Raap AK, Cornel-
D. W. H a m m o n d et al.
isse CJ (1991): Improved interpretation of complex chromosomal rearrangments by combined GTG banding and in situ suppression hybridization using chromosomal-specific libraries and cosmid probes. Genes Chrom Cancer 3:239-248.
ABSTRACT: We describe cytogenetic analyses of cells derived from 40 non-Hodgkins lymphoma (NHL)
node biopsies, 23 of which were from patients who had not been treated before biopsy. We noted that the chromosomes most frequently gained were X (32%), 12 (27%), and 3 (24%). Monosomies were much less common; loss of chromosome 13 (13.5%) was most frequent. Structural abnormalities primarily involved chromosomes 14 (70%), 1 (40.5%), 18 (38%), 6 (35%), and 17 (22%). Low- and high-grade disease showed similar patterns of structural changes; however, a markedly greater number of chromosome gains were associated with low-grade disease. Biopsy samples from patients who had previously been treated showed an increased frequency of structural abnormalities, as well as a significantly larger n u m b e r of chromosome gains. The importance of these observations, particularly with regard to possible oncogene involvement in lymphoma evolution, is discussed.
INTRODUCTION
MATERIALS A N D METHODS
Cytogenetic analyses of tumor cells have previously indicated that in m a n y cases a close relationship exists between specific c h r o m o s o m a l abnormalities and particular types of malignancy. This has become even more relevant in recent years w i t h the correlation of m a n y of these abnormalities to activation or deletion of oncogenes. Although some c h r o m o s o m a l changes occur very frequently, m a n y others occur more sporadically. In an attempt to determine the true significance of the latter, m a n y samples must be analyzed. In non-Hodgkins l y m p h o m a (NHL), several such studies have been p u b l i s h e d from centers in the United States [1-4], Canada [5], S w e d e n [6], France [7], the U.S.S.R. [8], Australia [9], and Japan [10]. Studies have also analyzed data gathered from several countries [11]. These investigations have been able to identify a number of nonr a n d o m abnormalities but have also raised more questions, not least that regarding regional variation in karyotypes [11, 12]. We report 40 karyotypes of cells isolated from NHL biopsies, a series we believe to be the largest yet reported from the United Kingdom.
Patients
From the Department of Clinical Oncology, Royal Hallamshire Hospital, (D. W. H., M. A., B. W. H., M. H. G.), the Centre for Human Genetics, (D. W. H., A. M. P.), and the Department of Pathology, University Medical School, Sheffield (J. R. G.), England. Address reprint requests to: Dr. Malcolm H. Goyns, Department of Clinical Oncology, Royal Hallamshire Hospital, Sheffield, $10 2JF, England. Received November I, 1991; accepted January 22, 1992. © 1992 Elsevier Science Publishing Co., Inc.
655 Avenue of the Americas, New York. NY 10010
Between 1989 and 1991, a consecutive series of 40 node biopsies, obtained from 37 white NHL patients, were studied cytogenetically. Twenty-three of the patients in this study were not treated before biopsy, but 17 of the s a m p l e s represent relapse cases who had been diagnosed p r e v i o u s l y and who had undergone some form of treatment. The diagnostic classification system used in this s t u d y was the updated Kiel classification [13]. Clinical details for each of the patients, relating to staging of the disease at presentation, types of treatment before biopsy, and diagnosis are s u m m a r i z e d in Table 1. Cytogenetic Methods
L y m p h node biopsy material was collected in Hams F10 medium, buffered with 25 mM HEPES. The tissue s a m p l e was cut with scissors to obtain cell suspensions. The latter were used to set up three cultures in p r e w a r m e d Hams F10 m e d i u m s u p p l e m e n t e d with 10% (vol/vol) p o o l e d h u m a n serum. Culture 1 had 0.1 ~g/ml Colcemid a d d e d before being incubated at 37°C for 30 minutes. Culture 2 had 0.01 ~g/ml Colcemid a d d e d before being incubated at 37°C overnight. Culture 3 was incubated overnight at 37°C and then had 0.1 /zg/ml Colcemid added, before being incubated for another hour. In all cases, the cells were centrifuged, r e s u s p e n d e d in 0,075 M KCI for 10 m i n u t e s at 37°C, and then centrifuged again before being fixed in 3 : 1 methan o l : a c e t i c acid. The cells were d r o p p e d onto glass slides (previously cleaned in Decon and stored in distilled water 31 Cancer Genet Cytogenet 61:31 38 (1992) 0165-46o8/92/$05.0o
32
Table 1
Patient
D . W . H a m m o n d et al.
Clinical details of patients Treatment Staging at before presentation biopsy
L001 L002 L003 L004 L005 L006 L007 L008 L009 L010A L010B L011 L012 L013 L014 L015 L016 L017 L018 L019 L020A L020B L021 L022 L023 L024 L025 L026A L026B L027 L028 L029 L030 L031 L032 L033 L034 L035 L036 L037
IIA IVA IVB IA IA [VA IA IIIA IA IIIB IA IVA IVA IVA IVA IVA IIIA IIIA IIA 1VA -IVB IIIA IIA ND IIIA IVA -IVA IVB IIIA IVB ND IIA IVB IVA IIIB IA IA
NT OC NT NT NT CC NT NT RT CC OCP OC OCP NT NT NT NT CC NT NT OC OCP NT OC NT NT RT NT NT NT NT OC NT NT OC NT OC CC OC NT
Diagnosis B-cell high-grade centroblastic B-cell low-grade follicular B-cell high-grade centroblastic B-cell low-grade follicular B-cell low-grade follicular B-cell high-grade centroblastic B-cell low-grade follicular B-cell low-grade follicular B-cell low-grade follicular B-cell low-grade follicular B-cell high-grade centroblastic B-cell low-grade centrocytic B-cell low-grade lymphocytic B-cell low-grade follicular B-cell low-grade follicular B-cell low-grade lymphocytic B-cell low-grade lymphocytic B-cell high-grade centroblastic B-cell low-grade lymphocytic T-cell low-grade T zone B-cell low-grade lymphocytic ~13-celllow-grade lymphocytic B-cell high-grade centroblastic B-cell low-grade follicular B-cell low-grade follicular B-cell high-grade immunoblastic B-cell high-grade centroblastic B-ceil low-grade lymphocytic B-cell low-grade lymphocytic B-cell low-grade follicular B-cell high-grade centroblastic B-cell high-grade centroblastic B-cell high-grade centroblastic B-cell low-grade follicular B-cell low-grade follicular T-cell high-grade immunoblastic B-cell low-grade follicular B-cell high-grade immunoblastic B-ceil low-grade follicular B-cell low-grade follicular
Abbreviations: CC, intravenous administration of combination chemother-
apy; ND, not determined; NT, no treatment; OC, oral administration of chlorambucil; OCP. oral administration of chlorambucil and prednisolone; RT, radiotherapy.
at 4°C) blotted, air-dried, and aged at 80°C. The chromosomes were G-banded with trypsin and Leishman stained [14]. Forty-one node samples from 38 patients were investigated in this way, of w h i c h 40 yielded analysable metaphases, a success rate of almost 100%. The karyotypes were described according to the International System for H u m a n Cytogenetic N o m e n c l a t u r e (ISCN) [15].
RESULTS The karyotypes of cells isolated from each of the node biopsies, the n u m b e r of cells e x a m i n e d and the proportion of normal cells observed are s u m m a r i z e d in Table 2. Only three of the specimens analyzed exhibited exclusively normal karyotypes. The r e m a in d e r contained cells with a variety of numerical and/or structural abnormalities. In about
half of the samples, a m i x of normal and abnormal cells was observed. Figure 1 shows the percentage of cases in w h i c h a gain or loss of each of the c h r o m o s o m e s was observed. The most frequently gained c h r o m o s o m e was X (32%), w h i c h was observed in both males (25%) and females (33%). This was followed in frequency by trisomy of c h r o m o s o m e s 12 (27%), 3 (24%), 7 (16%), and 8 (16%). M o n o s o m y was far less c o m m o n , with the most frequent loss i n v o l v i n g chrom o s o m e 13 (13.5%). With the e x c e p t i o n of L028 and L032, w h i c h both exhibited very c o m p l e x karyotypes w i t h several marker c h r o m o s o m e s , loss of a c h r o m o s o m e 11, 13, or 17 appeared to be m u t u a l l y exclusive. As a result, a quarter of the samples studied s h o w e d a loss of one of these chromosomes. Figure 2 summarizes the percentage of cases in w h i c h structural abnormalities were observed. With the exception of c h r o m o s o m e s 21 and Y, structural abnormalities inv o l v e d all chromosomes. The most frequently affected c h r o m o s o m e was 14 (70%), w h i c h in a p p r o x i m a t e l y half of these samples was i n v o l v e d in the t(14;18)(q32;q21). This correlated exactly with the presence of the reciprocal derivative c h r o m o s o m e s 1 8 q - . In some cases (11%), however, an apparently typical 1 4 q + derivative c h r o m o s o m e was observed, but the reciprocal 1 8 q - was absent. Several abnormalities were derived from c h r o m o s o m e 1 (40.5%), w h i c h in many cases appeared to i n v o l v e bands p 3 2 - 3 6 or q21. A b n o r m a l c h r o m o s o m e s 6 were observed in 35% of cases and most exhibited an altered q arm, often as a result of a deletion. Other c o m m o n l y rearranged c h r o m o s o m e s were 9 (22%), 17 (22%), and 3 (19%). In three cases (LOIOB, L020B, L026B), we were able to obtain second biopsy samples for analysis. The data from these were not i n c l u d e d in the analyses s h o w n in Figs. 1-4. Such data are of interest because of the possible insight they may provide into the e v o l u t i o n of the disease. In two cases (L020 and L026) there was little change in the disease or in the respective karyotypes. In the third case, however, the disease had transformed from low-grade (LO10A) to high-grade (LO10B), and this was paralleled by very significant changes in the karyotype. This series contained both high- and low-grade l ymphomas (Table 1), allowing c o m p a r i s o n b e t w e e n the karyotypes of these two classes of NHL. Because cases of loss of ch r o m o so m es were relatively few they have not been considered; however, n u m e r i c a l gains are s u m m a r i z e d in Fig. 3, w h i c h shows a remarkable difference b et w e e n lowand high-grade NHL, for most of c h r o m o s o m e gains appeared to be concentrated in the low-grade samples, although additional c h r o m o s o m e s 3 and X occurred at similar frequencies in both groups. Comparison of structural abnormalities s h o w e d a similar pattern in both groups, with only a slightly higher frequency in the low-grade samples. The series could also be split into a p p r o x i m a t e l y two equal-sized groups on the basis of treatment before biopsy. Again there were relatively few c h r o m o s o m e losses to analyze, but n u m er i cal gains are s u m m a r i z e d in Fig. 4. The samples from patients w h o had previously been treated, exhibited an increased frequency of gains of all chromo-
33
Table 2
Karyotypes of cells from NHL node biopsies
Patient
No. of cells examined
Percentage normal
L001
25
0
LO02 L003
29 34
0 0
LO04
49
L005
40
27
L006 L007
22 28
18
L008
18
0
LO09
24
12
L010A
25
0
L010B
18
0
L011
28
18
L012
28
18
L013 L014
22 22
0 18
L015 L016
23 24
0 4
L017
31
7
L018 L019
32 35
100 6
L020A
27
4
LO20B L021
17 27
0 15
L022
24
0
25
0
Karyotype
47,XY, + 3,dup(1)(qllq44),del(2){q21),del(9)(q21}, der(11}t(11;?)(q23;?) 46,XX,inv(9)(p13q22},t(14;18)(q32;q21),der(16)t(16;?)(p13.3;?) 46,XY, + X,der(3)t(3;?)(q13;?),der(6)t(6;?)(q13;?), - 8, del(14)(q22q24),del(17)(p13) 46,XY 46,XY,del(5)(p14),der(12)t(12;?)(p11;?),der(15)t(15;?)(q22;?} 46,XX 48,XX,+ 8,+ 12 4 9 , X X , + X , + 8 , + 12 46,XX,del(14)(q31),t(20;22)(q13.3;q12),der(14)t(14;?)(q32;?) 46,XY 94,XXYY, + X x 2,der(1)t(1;?)(p13;?),der(2)t(2;?)(p21;?) x 2,der(3)t(3;?) (q13;?), - 4,del(6)(q16q22), + 12 x 2, - 17,t(14;18)(q32;q21) x 2 48,XX, + 3,del(6)(p21), + del(6)(q21),t(10;13)(q22;q12),del(14)(q31), del(17)(p11),der(22)t(22;?)(q13;?) 46,XX 62,XX, + der(1)t(1;?)(p11 ;?), + 2, + ins(3;?)(q21;?) x 2, + del(5)(q31), + del(7)(q32) x 2, + 8, + 9,del(10)(q22),der(10)t(10;?)(q23;?), + 11, + 12, + 13,t(14;18)(q32;q21), + der(14)t(14;?)(q32;?), + 19, + 20, + 21 92,XXXX, + X,der(1}t(1;?)(p36;?), + del(3)(p21}, der(3)t(3;?)(p13;?),del(6)(q15q21),t[del(9)(p22);13](q21;q12), dup(12)(q13q22),der(13)t[del(9)(p22);13](q21;q12), t(14;18)(q32;q21),- 1 5 , - 16,der(16}t(16;?)(q12;?),der(19}t(19;?)(p11;?) 48,XX, + der(X)t(X;?)(p22;?),der(1)t(1;?)(p36;?),der(3},t(3;?)(q21;?), del(6)(q15q21), + 7,dup(12)(q13q22),der(13)t[del(9)(p22);13] (q21;q12),t(14;18)(q32;q21),der(19)t(19;?)(q13.4;?) 46,XY 49,XY, + 8, + 9,der(14)t(14;?)(q32;?), + 18 49,XY,ins(1)(q21q257), + 8, + 9,der(14)t(14;?)(q32;?), + 18 49,XY, + 8, + i(9q),der(14)t(14;?)(q32;?), + 18 46,XY 48,XY,der(1)t(1;?)(p32;?), + 7, - 11,del(14)(q24),del(15)(q24), del(20(q13), + 2mar 4 5 , X , - X , + 1 2 , - 14 46,XX 46,XX,t(6;8)(p21;q24},t(14;18)(q32;q21) 47,XX,t(6;8)(p21;q24), + 8,t(14;18)(q32;q21) 48,XX,t(14;18)(q32;q21), + 18, + der(18)t(18;?)(q21;?) 46,XX 47,XX, + 12,del(14)(q22} 46,XY 47,XY, + X,der(6)t(6;?)(q23;?),ins(7;?)(p13;?),t(9;17)(q11;p11), + 1 2 , - 13,t(14;18)(q32;q21),- 15, + 18 46,XY 46,XY 46,XY, - 5, - 9,der(10)t(10;?)(p11;?),der(11)t(11;?)(q21;?), der(16)t(1;16)(q21;p11), + 2mar. 46,XX 46,XX,der(11}t(ll;?)(q13;?) 46,XX,der(11)t(11;?)(q13;?) 46,XY 47,XY,der(1)t(1;?)(q21;?),der(5)t(5;?)(p13;?),- 8 , - 9 , - 10, - 1 1 , - 13,del(13q31),der(14)t(14;?)(q24;?), + der(17)t(17;?) (p12;?), + 22, + 4mar 50,XX, + X,der(1}t(1;9)(p32;q12),der(5)t(5;?)(q13;?), + der(5}t(5;?)(q13;?),der(6)t(6;?;5)(q13;?;q13), - 8, + 12,der(14)t(14;?)(q32;?) x 2,der(15)t(1;15)(q21;q22), + 18, + mar
(continued)
34
D.W. Hammond
Table 2
K a r y o t y p e s of cells f r o m N H L n o d e b i o p s i e s
Patient
No. of cells examined
Percentage normal
L023
29
14
L024 L025
20 23
0 22
L026A L026B L027
25 21 19
0 0 11
L028
29
14
L029
35
0
L030 L031
23 36
0 17
L032
32
50
L033 L034
59 24
97 13
L035 L036
37 33
100 9
L037
21
19
et al.
(Continued) Karyotype
46,XX 53-55,XX, + X,dup(1)(q21q32),der(1)t(1;?)(p32:?), der(2)t(2;?)(p21;?), + 3, + 5,i(6p), + 7, + der(8)t(8;?)(p11;?), + 11,t(13q15q),t(14;18)(q32;q21),- 15,der(18)t(18;?)(q21;?), + 21, + 2r 47,XY,der(1)t(1;?)(q42;?},del(2)(q23),t(8;14)(q24;q32), + 21 46,XX 51,XX,dup(1)(qllq25),der(2}t(2;?)(p23;?), + 3, + 3, der(22)t(22;?)(q13;?), + 3mar 46,XY, + 1 2 , - 13,der(17)t(17;?)(q21;?) x 2 46,XY, + 1 2 , - 13,der(17)t(17;?)(q21;?) x 2 46,XX 48,XX, + 5,i(6p), + 12,t(14;18)(q32;q21), - 18, + mar 46,XX 43-46,X,der(X)t(X;?)(p22;?),der(1)t(1;?)(p32;?), + 3,der(4)t(4;?)(q21;?), - 6,der(7)t(7;?)(p21 ;?), der(8)t(8;?)(p21;?),- 1 0 , - 13,der(16)t(16;?)(p12;?), 17,der(18)t(18;?)(q23;?),der(20)t(X;20;?)(q13,q13;?),- 22, + 3mar 88,XXYY, - 5,del(6)(q23q26), - 9, - 10, - 13,t(14;18)(q32;q21), - 16, del(16)(q22), - 20, + 2mar 49,XY, + X, + 12, + mar 46,XX 46,XX,der(14)t(14;?)(q32;?) 46,XX,der(3)t(3;?}(q27;?),der(14}t(14;?)(q32;?) 46,XX,der(14)t(14;?)(q32;?),del(17)(p13) 46,X,del(X)(q28),del(2)(p23),der(14)t(14;?)(q32;?),del(20)(p12) 46,XY 79-82,XXY,- Y,i(1q),der(1)(1;?)(p36;?) × 2,der(1)(1;?)(p36;?), der(2)t(2;?)(p13;?),i(3p), + der(3)t(3;?)(p13;?), - 4, del(4)(q12q25), + del(6)(q22q23) × 2,del(7)(q22) × 2, 8,der(8)t(8;?)(p11;?),- 9,der(9)t(9;?)(q34;?),- 11 x 2, - 12,der(12)t(12;?)(p13;?),- 13,der(14)t(14;?)(q22;?), der(16)t(16;?)(q11;?) × 2, + i n v ( 1 6 ) ( q l l p 1 2 ) , - 1 8 , - 1 9 , - 21, + 3mar 46,XX 46,XX 49,XX, +X, ÷ 7, + 8,der(6)t(6;?)(q23;?) x 2, t(14;18)(q32 ;q21),der(17)t(17 ;?)(q21 ;?) 46,XY 46,XX 74,XX, + X × 2, + der(1)t(1;?)(p13;?), + 2, + der(3)t(3;?)(q27;?), + 4, + 5, + 6, + der(6)t(6;?)(q23;?), + 7, + 8, + 9, + 11, + 13, + 16, t(14;18)(q32;q21) × 2 , - 19, + 20, + del(22)(q11), + 11mar 46,XX 46,XX,t(14;18)(q32;q21) 46,XX,t(14;18)(q32;q21),t(9;19)(p13;q13.1)
s o m e s , w i t h t h e e x c e p t i o n of c h r o m o s o m e 12 a n d , to a m u c h l e s s e r e x t e n t , c h r o m o s o m e s 21 a n d 22. O c c u r r e n c e of structural changes showed a similar pattern in both groups, a l t h o u g h t h e y a p p e a r e d to b e m o r e c o m m o n i n t h e t r e a t e d group. Low-grade samples in particular showed more struct u r a l a b n o r m a l i t i e s after t r e a t m e n t t h a n t h e h i g h - g r a d e group.
D I S C U S S I O N
W e r e p o r t t h e c y t o g e n e t i c a n a l y s e s of a series of 40 NHL n o d e b i o p s i e s o b t a i n e d f r o m U.K. p a t i e n t s . T h i s g r o u p w a s c o m p o s e d of b o t h l o w - a n d h i g h - g r a d e N H L s a m p l e s , a n d
a p p r o x i m a t e l y e q u a l n u m b e r s of n o n t r e a t e d a n d p r e v i o u s l y treated patients. The most striking finding was the much g r e a t e r p r e v a l e n c e of n u m e r i c a l g a i n s i n t h e l o w - g r a d e N H L s a m p l e s . T w i c e as m a n y of t h e s e s a m p l e s e x h i b i t e d trisom i e s of s e v e r a l c h r o m o s o m e s t h a n d i d t h o s e i n t h e h i g h g r a d e g r o u p . F u r t h e r m o r e , t r i s o m i e s of a l m o s t all t h e c h r o mosomes were observed in low-grade samples, whereas o n l y s e v e n of t h e c h r o m o s o m e s w e r e i n v o l v e d i n t r i s o m i e s in the high-grade group. To our knowledge, this observation h a s n o t b e e n p r e v i o u s l y d o c u m e n t e d . It m a y b e a n i n d i c a t i o n of a m o r e g e n e r a l b r e a k d o w n i n t h e m a c h i n e r y of cell r e p l i c a t i o n i n l o w - g r a d e d i s e a s e or, as t h i s effect h a s n o t b e e n n o t e d before, it m a y r e p r e s e n t a n e x a m p l e of r e g i o n a l
NHL Karyotypes
35
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1 ' 2 ' 3 ' 4 ' 5 ' 6 ' 7 ' 8 ' 9 ' 1 0 ' 1 1 ' 1 2 ' 1 3 ' 1 4 ' 1 5 ' 1 6 ' 1 7 ' 1 8 ' 1 9 ' 2 0 ' 2 1 ' 2 2 ' X Y;
Figure 1 Analysis of numerical changes in NHL node biopsy samples. (a) Percentage of cases exhibiting chromosome gains; (b) percentage of cases exhibiting chromosome losses.
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Figure 3 Comparison of numerical gains of chromosomes in high- and low-grade NHL. (a) Percentage of highgrade cases exhibiting chromosome gains; (b) percentage of low-grade cases exhibiting chromosome gains.
36
D . W . H a m m o n d et al.
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Figure 4 Comparison of numerical gains of chromosomes in biopsy samples from nontreated and treated NHL patients. (a) Percentage of nontreated cases exhibiting numerical gains; (b) percentage of treated cases exhibiting numerical gains.
variation [11, 12]. Further analysis of our data, however, s h o w e d that trisomies a p p e a r e d to be more c o m m o n in low-grade disease samples from patients who had been p r e v i o u s l y treated than in similarly treated high-grade sampies. The effect of treatment m a y therefore have had a m u c h greater effect on the c h r o m o s o m e s in low-grade than in high-grade NHL. The most c o m m o n n u m e r i c a l c h r o m o s o m e change observed was the presence of an a d d i t i o n a l c h r o m o s o m e X, present at a higher frequency than in previously reported series, in w h i c h the frequency varied between 3 and 22.5% [3, 4, 8]. The significance of an extra c h r o m o s o m e X is unclear, although studies of other types of malignancy have i n d i c a t e d that a d d i t i o n a l X chromosomes are active in neoplastic cells [16, 17]. In some NHL cases, an extra chromosome X has been reported to be the only identifiable abnorm a l i t y [18]. Trisomies involving c h r o m o s o m e s 3 and 12 were observed at frequencies similar to those previously reported [3, 4, 8, 11]. Our data do not support the suggestions that + 3 is associated with high-grade l y m p h o m a s [5, 8], or that + 12 is associated with l y m p h o c y t i c [18] or diffuse large cell l y m p h o m a [3, 19]. In our series, + 12 was twice as c o m m o n in low-grade NHL but was not particularly associated with the l y m p h o c y t i c subtype. Alone among the c h r o m o s o m e gains, + 12 was more frequent in patients w h o had not been treated; even though low- and high-grade samples were evenly distributed between the treated and nontreated groups. Loss of a whole c h r o m o s o m e d i d not appear to be a frequent occurrence; however, further analysis demonstrated that w h e n loss of c h r o m o s o m e s 11, 13, or 17 was observed, u s u a l l y only one of these c h r o m o s o m e s was lost in any particular sample. This is n o t e w o r t h y because m a n y studies of other malignancies have demonstrated the pres-
ence of t u m o r supressor genes on these chromosomes. Chromosome 13 carries the RB-1 gene [20, 21], c h r o m o s o m e 17 the p53 gene [22], and c h r o m o s o m e 11 the W i l m s ' tumor gene [23]. That loss of these c h r o m o s o m e s occurs in a quarter of the cases in our study suggests that further m o l e c u l a r biology studies of the i m p l i c a t e d t u m o r s u p p r e s s o r genes may be informative. In comparison, deletions of parts of c h r o m o s o m e s were more frequent. In particular, c h r o m o s o m e 6 was often observed to have part or all of its q arm deleted. If translocation breakpoints are also i n c l u d e d , 27% of all cases exhibited deletion or rearrangements of 6q21-25. Similar findings have been d o c u m e n t e d p r e v i o u s l y [3, 8, 11]. We found no evidence to s u p p o r t the suggestions that these abnormalities were p r i m a r i l y associated either w i t h p r e v i o u s treatment [19] or i m m u n o b l a s t i c l y m p h o m a [5]. Indeed, our data suggested a more c o m m o n association of 6q abnormalities in low-grade disease. Structural abnormalities of 6q have been observed in a w i d e range of m a l i g n a n t cells [18], and several protooncogenes have been localized to this region [24]. The most extensively s t u d i e d of these is MYB which, as a result of the 6 q - deletions, has been observed to be altered in a n u m b e r of l y m p h o m a cell lines [25]. Whether MYB is the target gene associated w i t h the 6q abnormalities is still uncertain, however. From the c h r o m o s o m a l abnormalities observed in this series, several other oncogenes c o u l d be i m p l i c a t e d in the evolution of NHL; e.g., we noted l p 3 2 - 3 6 (L-MYC), 17p13 (p53), and 19q13 (BCL-3) breakpoints in several cases. In only one case, however, d i d we observe a translocation involving 11q13 (BCL-1). The most c o m m o n c h r o m o s o m a l a b n o r m a l i t y we detected was the t(14;18)(q32;q21}, w i t h frequencies of 59% in follicular low-grade and 17% in high-grade NHL. These
NHL Karyotypes
figures are in agreement w i t h results of some previously p u b l i s h e d studies [3, 5], but lower than those of others [4, 8]. This translocation appears to be the most c o m m o n m e c h a n i s m by w h i c h the BCL-2 gene is activated [26]. Although the frequency of the translocation in our series was not as high as reported in some others [4, 8], we did note that in 11% of the cases an apparently typical 14q + derivative c h r o m o s o m e occured w i t h o u t a reciprocal 1 8 q - . The latter may have been lost or masked by further translocation events in these cells, w h i c h w o u l d mean that the frequency of the t(14;18) may actually be higher in our series. This situation highlights the problems of analyzing c o m p l e x karyotypes, such as those w h i c h occur in NHL, w h e n banding patterns alone are used. Further advances in understanding abnormal c h r o m o s o m e structure may therefore require application of n e w m e t h o d s such as nonradioactive in situ hybridization of DNA probes [27].
This work was supported by the Yorkshire Cancer Research Campaign.
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