Nonrandom Cytogenetic Changes in Leiomyomas of the Female Genitourinary Tract A Report of 35 Cases Marion Kiechle-Schwarz, Chandrika Sreekantaiah, Carol S. Berger, Steve Pedron, Michael T. Medchill, Urvashi Surti, and Avery A. Sandberg
ABSTRACT: Cytagenetic analysis of short-term cultures fl'om 35 leiomyomas of the female genitourinary tract showed abnormal karyotypes in 14 cases. In 11 o~ 14 aberrant tumors, normal cells were also observed. Structural changes were most frequent, resulting in modal chromosome numbers in the diploid range. Our data confirm preferential breakpoint (:lusters at 7q, 12q14-15, and 14q23 24, mainly resulting from consistent, specific chromosome rearrangements such as tI t2.14)[q14 15;q23-24) and del(7)(q21) or del(7)(q22q32). Tagether with previausly published cases, we describe trisamy 12, ring chromosomes, and manosomy 22 as new additianal recurrenl findings in myomas. Statistical analyses of possible caherencies between tumor karyotype (abnormal versus normal) and clinicopathologic data, as well as age of tbe patie.nts, menopausal status, and tumor size showed no correlatians.
INTRODUCTION T h e l o n g - m a i n t a i n e d g e n e r a l c o n c e p t [1] t h a t a n a b n o r m a l c h r o n l o s o n l e p a t t e r n is r e l a t e d to a m a l i g n a n t or p r e m a l i g n a n t state a n d a n o r m a l d i p l o i d k a r y o t y p e to b e n i g n s t a t e s m u s t be r e v i s e d . A n i n c r e a s i n g n u m b e r of r e c u r r e n t c l o n a l c h r o m o s o m e c h a n g e s h a v e b e e n d e t e c t e d i n b e n i g n t u m o r s , s u c h as l i p o m a s [2], p l e o m o r p h i c a d e n o m a s [3], a n d m e n i n g i o m a s [4]. R e c e n t l y , l e i o m y o m a s w e r e a d d e d to t h i s list [ 5 - 2 1 ] . M o r e over, t h e c h r o m o s o m e d e v i a t i o n s f o u n d in s o m e l i p o m a s a n d m y o n l a s w e r e as c o m p l e x as t h o s e u s u a l l y s e e n in a d v a n c e d c a n c e r s [19, 22]. P a r a l l e l c y t o g e n e t i c s t u d i e s of b e n i g n a n d m a l i g n a n t m e s e n c h y m a l n e o p l a s m s o r i g i n a t i n g f r o m fat a n d s m o o t h m u s c l e t i s s u e s h a v e s h o w n a l t e r a t i o n s in t h e s a m e c h r o m o s o m e b a n d s [231. T h u s , c y t o g e n e t i c i n v e s t i g a t i o n s of b e n i g n n e o p l a s m s are e x p e c t e d to e l u c i d a t e o u r u n d e r s t a n d i n g of d i f f e r e n c e s b e t w e e n b e n i g n a n d m a l i g n a n t p r o l i f e r a t i o n a n d m i g h t p r o v i d e i n t e r e s t i n g i n f o r m a t i o n a b o u t t h e role of c e r t a i n c h r o m o s o m e c h a n g e s in t h e e v o l u t i o n of m a l i g n a n t p h e n o t y p e s . C y t o g e n e t i c s t u d i e s of u t e r i n e l e i o m y o m a s h a v e s h o w n r e c u r r e n t , c l o n a l c h r o m o From tile Cancer Center of the Southwest Biomedical Research Institute of Genetrix, Scottsdale, Arizona (M. K.-S., C. S., C. S. B., A, A. S.) Department of Gynecology and Obstetrics, The University Hospital, Freiburg, F. R. G. (M. K.-S.], the Maricopa Medical Center Phoenix, Arizona (S. P., M. T. M.), and tbe Magee Womens Hospital Department of Pathology, Pittsburgh. Pennsylvania (tl. S.). Address reprint requests to: A. A. Sandberg, M.D., The Cancer Center of the Southwest Biomedi(:al Institute af Genetrix, Scottsdale, AZ 85251. Received June 12, 1990; accepted September 18, 1990.
125 © 1991 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet 53:125 136 (1991) 0165-4608/91/$03.50
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M. Kiecble-Schwarz et al. somal alterations, affecting particularly chromosomes 7, 12, and 14 15-9, 11 14, 16 18, 29]: some of these alterations had been suggested to be specific rearrangements. We report detailed cytogenetic observations of 35 myomas from 33 patients and five specimens of adjacent normal myometrium. We discuss our findings and their significan(:e with regard to previously published cytogenetic studies on leiomyomas and other benign neoplasms. We also present possible correlations between clinicopathologic parameters (age, menopausal status, and tumor size) and cytogenetic findings.
MATERIALS A N D METHODS
We cytogenetica]ly analyzed short-term cultures of fresh samples of uterine tumors and c o r r e s p o n d i n g normal m y o m e t r i u m obtained at surgery. All tumors were macroscopically typical leiomyomas. Histologically, they were all benign, with no atypical cells showing nuclear p l e o m o r p h i s m or increased mitotic activity. Some pertinent clinicopathological data of the patients are summarized in Table 1. Tissue cultures and chromosome preparations were made as previously described [21]; this involved disaggregation of the tumors overnight in collagenase (200 U/lnl) and culture in flasks and on cnverslips for a period of 4 - 2 2 days. Harvest time was i n d i v i d u a l i z e d for each flask and coverslip and included Coh:emid exposure, hypotonic shock, and fixation. Metaphases were G-banded using trypsin-(;iemsa [24]. Karyotypes were constructed ac(:ording to the International System for Human Cytogenetic Nomenclature [25]. Statistical significance was calculated by Wih:oxon-MannWhitney II test, as described by Sachs 126]. RESULTS
Thirty-five myoinas from 33 patients and five specimens of normal myometriunl were studied cytogenetically. The detailed cytogenetic and tissue culture data are shown in Tables 2-4. At least five and up to 30 metaphases were analyzed for each sample. Chromosome analysis of cells cultured from adjacent normal myometrial tissue showed a normal karyotype in three cases (N5, N10, N l l ) . Interestingly, in case N14 two cells with trisomy 12 as the only abnormality were found in addition to normal cells. Case N15 failed to grow in culture (Table 2). Eighteen of 35 tumors showed an a p p a r e n t l y normal female karyotype whereas in 14 cases an abnormal chromosome c o m p l e m e n t was found. Three tumors failed to grow in culture. Table 3 shows the detailed cytogenetic data of 18 leiomyomas I c a s e s M 1 5 M32) with a predoiuinantly normal karyotype. Case M30 had a relatively high number of tetraploid cells {20%). Two myomas (M2 and M21) had one cell each with a terminal deletion affecting the long arln of cbrolnosome 7 and the short arm of chromosome 1, respectively, However, with regard to cytogenetic findings in myomas discussed below, the breakpoint 7q22-q23 is of interest. Table 4 shows the detailed cytogenetic results of 14 tumors with abnormal karyotypes. Most of them (M1, M2, M7 Mg, M l l M14) showed cells with a normal karyotype in a d d i t i o n to the abnormal cell line. Three lnyomas (M3, M4, M5) contained only abnormal cells. Both case M6 and case M10 had two abnormal cell clones. The various abnormalities (except M13 and the previously published case M14) are shown in Figures 1 4 b y partial karyotypes. The modal c h r o m o s o m e munber of all deviant cell lines was in the diploid range. Structural rearrangements included deletions, transhications, insertions, and ring c h r o m o s o m e s and occurred in a significantly higher frequency than numerical changes. Loss or gain of whole chromosomes was nbserved in four cases (M2, M3, M6, M l l ) : Case M6 showed trisomy 12 as the only numerical change. Cases M2, M3,
12 7
Cytogenetics of 35 Leiomyomas Table 1
Pertinent clinical data of patients with m y o m a s
Case
Age
1 2 3 4 5" 6 7 8 9 1if' 11 ~ 12 13 14 ~'J' 15 ~' 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3t) 31 32 33 34
35 39 46 46 32 63 34 48 29 39 49 33 32 56 34 45 41 42 51 51 54 42
Pre Pre [)re Pre Pre Post Pre Pre Pre Pre Pre Pre Pre Post Pre Pre Pre .Pre . .
33 37 43 59 46 43 43 4:] 44 61 41 45
Pre Pre Pre Post Pre Pre Pre Pre
35
Menopause
T u m o r size (cm)
Karyotype
10.2 2.0 6.0 20.2 7.5 3.O 4.0 5.0 6.5 3.0 4.(I
Abnormal/normal Abnormal/normal All abnormal All abnormal All abnormal Abnormal/normal Abnormal/normal Abnormal/normal Abnormal/normal Abnormal/normal Abnormal/normal Abnormal/normal Abnormal/normal Abnormal/normal All normal All normal All normal All normal All normal All normal All normal All normal All normal All normal All normal All normal All normal All normal All normal All normal All normal All normal No results No results No results
12.0 11.0 9.5 7.0 7.0 8.0 2.5 .
.
. 3.8 7.5
Post Pre Pre
3.0 11.0 3.0 3.0 1.8 8.5 5.5 10.5 -13.0 6.0
Cases 12 and 24 are the same patient, as are cases 30 and 31. " Corresponding normal myomelrium is analyzed: see Table 2. ~'Previously published.
a n d M l l s h o w e d m o n o s o m y 22 b e s i d e s o t h e r s t r u c t u r a l a b n o r m a l i t i e s : M11 a l s o s h o w e d m o n o s o m y 6. S t r u c t u r a l r e a r r a n g e m e n t s i n v o l v e d d e l e t i o n s a n d r e s u l t e d ill p a r t i a l l o s s of c h r o m o s o m e s e g m e n t s in e i g h t m y o m a s (M2, M 6 - 1 1 , M14). M o s t f r e q u e n t l y , p a r t s of 7q w e r e l o s t (M6, M 7 , M 1 0 ) , a n d it w a s t h e o n l y r e a r r a n g e m e n t in t w o t u m o r s . S e g m e n t s of 14q, 6q, 20q, 2p, a n d 10q w e r e d e l e t e d in o n e c a s e e a c h . T r a n s l o c a t i o n s t h a t m o s t c o m m o n l y i n v o l v e d c h r o m o s o m e s 12 a n d 14 w e r e p r e s e n t i n t h r e e t u m o r s (M1, M2, M3) as a r e c i p r o c a l e v e n t a n d i n d e p e n d e n t l y in c a s e s M4, M5, a n d M10. Ring c h r o m o s o m e s d e r i v e d f r o m c h r o m o s o m e 1 (M12) a n d u n d e f i n a b l e c h r o m o s o m e m a t e rial (M13) w e r e o b s e r v e d in t w o c a s e s . W i t h r e g a r d to i n v o l v e i n e n t of c h r o m o s o m e r e g i o n s a f f e c t e d by s t r u c t u r a l a b e r r a t i o n s in t h e 14 a b n o r m a l m y o m a s , t h e f o l l o w i n g o b s e r v a t i o n s w e r e m a d e : M o s t fre-
128
M. K i e c h l e - S c h w a r z et al.
Table 2
C h r o m o s o m a l f i n d i n g s in five s p e c i m e n s of n o r m a l m y o m e t r i u m
Case
Culture time (days)
No. of cells analyzed
Karyotype
N5 N10 N11 N14"
10 8 9-10 9-10
16 15 15 18 2
46,XX 46,XX 46,XX 46,XX 47,XX, + 12
N15
No growth
Abbreviations: N. normal myometrium. " Previously published.
q u e n t l y , b r e a k s o c c u r r e d at 1 2 q 1 4 - 1 5 (five cases), 1 4 q 2 3 - 2 4 (four cases), l p 3 6 (three cases), 6p21 (two cases), a n d 7 q 2 1 . 2 - 2 2 (two cases). F i g u r e 5 s h o w s t h e b r e a k p o i n t d i s t r i b u t i o n (37 b r e a k p o i n t s in all). M u l t i p l e m y o m a s w e r e a n a l y z e d f r o m t w o p a t i e n t s . T u m o r s M 3 0 a n d M31, d e r i v e d from one patient, both showed a normal female chromosome constitution, but M24 a n d M 1 2 , also f r o m o n e p a t i e n t , h a d d i f f e r e n t k a r y o t y p e s (M24:46,XX; M 1 3 : 4 6 , X X / 47,XX, + r). C o m p a r i s o n of t h e c y t o g e n e t i c a l l y a b n o r m a l m y o m a s v e r s u s t u n m r s w i t h a n o r m a l c h r o m o s o m e c o m p l e m e n t w i t h r e g a r d to c l i n i c a l f e a t u r e s (Table 1) s h o w e d n o c l i n i c o c y t o g e n e t i c c o r r e l a t i o n s . No c o r r e l a t i o n b e t w e e n n o r m a l or a b n o r m a l k a r y o t y p e a n d age or m e n o p a u s a l s t a t u s of t h e p a t i e n t s w a s o b s e r v e d . T h e r e a p p e a r e d to be a p o s i t i v e c o r r e l a t i o n b e t w e e n t u m o r size a n d a b n o r m a l k a r y o t y p e : h o w e v e r , n o s t a t i s t i c a l sig-
Table 3
D e t a i l e d c y t o g e n e t i c d a t a o n 18 l e i o m y o m a s w i t h a n a p p a r e n t l y normal female karyotype
Case
Culture time (days)
No. of cells analyzed
Karyotype
M15 M16 M17 M18 M19 M20 M21 M22 M23 M24 M25 M26 M27 M28 M29 M30 M31 M32
10-11 13 4-5 6 4 6 20-22 4-10 7-11 4-10 5 8-13 5-7 4 6-9 7-12 8-9 8-15 8-9
2(I 6 15 20 2(I/1 17 13/1 19/1 2(I 22 13 21 21 2(I 21/2 20/5 18 20
46,XX 46,XX 46,XX 46,XX 46,XX/92,XXXX (lqh +) 46,XX 46,XX/46,XX,del(7)(q22q23) 46.XX/46,XX,deI[1)(p13) 46,XX 46,XX 46,XX 46,XX 46,XX 46,XX 46,XX/92,XXXX 46,XX/92.XXXX 46,XX 46,XX
Abbreviation: M, leiomyoma.
129
C y t o g e n e t i c s of 35 L e i o m y o m a s
Table 4
D e t a i l e d c h r o m o s o m e r e a r r a n g e m e n t s in 14 l e i o m y o m a s of t h e f e m a l e g e n i t o u r i n a r y tract
Case
Culture time (days)
M1
3-5
M2
4
M3 M4
2-7 5
M5 M6
7-8 5-6
M7
5
M8
5-9
M9
5-8
M10
5
Mll
10 18
M12
6
M13
5-7
M14
4-5
Karyotype 46,XX,t(12;14)(q 15:q24) 20 46,XX - 2 45,XX,t(12;14)(q14:q23-24], 22 9 46,XX 6 45,XX,ins[inv(2)(qllq37);15](ql 1 :q12q26),t(12;14](q15:q24), - 22 19 46,XX,t(9;12)(p22;q15) 21 92,XXXX,t(9;12)(p22 ;q15),t(9;12)(p22:q15) 2 46,XX,t(2;12)(q36:q15) 21 46,XX = 7 47,XX,+ 12 5 46,XX,del(7)(q22q35] 4 6 cells with additional nonclonal changes 46,XX = 9 46,XX,del(7)(q21.2q31.2) - 2 46,XX 14 46,XX,del(6)(q23q25) 6 45,XX,del(1)(p34.1p36.1), - 3, + der(3)t(3:?)(p25;?), 6, + der(6)t(6;?) (p21.1;?),del(20)(q11.2q13.3),- 21 = 11 46,XX 9 (one cell: 46,XX,lp ) 46,XX 11 46,XX,t(6;14)(p21.3;q24) 4 45,XX, - 7, - 8, ÷ der(8)t(7;8)(p12;p23) 3 3 cells with additional nonclonal changes 46,XX 13 43,XX, - 1, + der(1)t(1:13)(p36.2 or p36.3;q12),del(2)(p21), 6 , - 1 3 , - 2 2 , - 6 3 cells with additional nonclonal changes 46,XX,r(1?) 11 46,XX = 9 46,XX = 27 47,XX,+r 3 46,XX,del(10)(q22q24) 12 46,XX - 8
n i f i c a n c e w a s o b t a i n e d w i t h W i l c o x o n - M a n n - W h i t n e y U test [26] w i t h regard to t u m o r size a n d k a r y o t y p e ( p v a l u e - 0.4).
DISCUSSION To date, 230 l e i o m y o m a s , i n c l u d i n g t h e c a s e s w e r e p o r t , h a v e b e e n s u c c e s s f u l l y karyotyped. Apparently normal female chromosome constitutions have been reported for 146 t u m o r s in c o n t r a s t to 84 c a s e s (36%) w i t h c l o n a l c h r o m o s o m e a b e r r a t i o n s [ 5 - 2 1 , 29]. In m o s t of t h e a b e r r a n t c a s e s , t h e m o d a l c h r o m o s o m e n u m b e r has b e e n in t h e d i p l o i d range. T h u s , s t r u c t u r a l c h r o m o s o m e c h a n g e s u n e q u i v o c a l l y r e p r e s e n t e d t h e m a j o r i t y of t h e o b s e r v e d d e v i a t i o n s . A c c o r d i n g to t h e a b o v e r e p o r t s a n d o u r o w n i n v e s t i g a t i o n s , c h r o m o s o m e breaks o c c u r in a n o n r a n d o m p a t t e r n a n d c l u s t e r to c h r o m o s o m e r e g i o n s 7 q 2 1 - 3 2 , 1 2 q 1 3 - 1 5 , a n d 1 4 q 2 1 - 2 4 (Fig. 5). A d d i t i o n a l c h r o m o s o m e b a n d s p r e f e r e n t i a l l y rea r r a n g e d are l p 3 6 , 2 p 2 4 - 2 5 , a n d 6p21. T h i s b r e a k p o i n t d i s t r i b u t i o n r e s u l t s
130
M. Kiechle-Schwarz et al.
MI 12
14
12
14
rd2 22
M3
i, 7 12
14
M4
15
Ir~*. 9
12
;[
P
22
-,el-
M5
B
12
2
Figure 1
Partial karyolyp(~s of (:as(~s MI to MS.
mainly from recurrently described rearraugements, namely t(12;14) (q13-15;q21-24),del{7)(q21) or del(7)(q21q32), and t(l:2)(p36;p24), shown in detail in Table 5. Deletion 7q and the 12;14 translocation are tbe most consistent findings in myomas and have been reported in 16 cases each. Their frequent occurrence even as sole abnormalities suggests that they are primary specific events playing an important role in myoma development and proliferation: these changes have been the basis for delineation of cytogenetic subgroups of myomas [12, 15]. However, several cases of leiomyomas showing two or more of these recurrent changes at the same time have been reported [17, and present study]. Although numerical changes are relatively rare in myomas, trisomy 12 has been
131
Cytogenetics of 35 Leiomyomas
a
b
M6
! ~!i~¸¸¸:i~
12
7
M7
7
M8
6 Figure 2
Partial karyotypes of cases M6, M7, and MS.
observed in 10 cases (Table 5). Because it was the sole abnormality in six cases, it is considered to constitute another primary specific aberration. In addition to the primary recurrent abnormalities, various structural and nnnmrical changes have been observed in some cases [101, reflecting clonal evolution within the tumor cell populations. Furthermore, some leiomyomas have nonrecurrent chmal abnormalities [11, 18, and present studyl, some showing very complex chromosome rearrangements [19] and affecting the same bands as a leiomyosarcoma 121]. In the present study of 35 leiomyomas, the frequency of normal female chromosome complement was 51% (18 of 35). Abnormal karyotypes were found in 40% {14 of 35), and the failure rate was < 10% [3 of 35). Similar results were obtained by other groups of investigators studying fewer cases, however [9, 18]. In our series, tumors with cytogenetically abnormal and normal cells (11 of 14), more than one abuormal clone (1 of 14), and only one abnormal cell line (3 of 14) were found. The relatively high n u m b e r of apparently normal cells coexisting with abnormal clones appears to represent a c o m m o n feature of benign tuinors, such as meningiomas [4I, pleomorphic
132
M. Kiechle-Schwarz et al.
= 1
6
i 3
6
14
20
21
8
7
6
22
MII
I
13
2
Figure 3 Partial karyotypes of the myomas with complex chromosome rearrangements (Mg, M10, Mll).
adenomas [3], lipomas [2], and leiomyomas [9, 11, 18]. For these tumors, the normal cell lines, either found alone or in combination with an abnormal clone, have been suggested to represent proliferating neoplastic cells. Detailed studies on tumors with normal stemlines showed that their cells possess a higher chromosome instability as c o m p a r e d with n o n n e o p l a s t i c cell populations, which resulted in formation of variant cells carrying a variety of nonclonal numerical and/or structural aberrations [9, 18]. Certain aberrations may lead to an increasing growth potential and may constitute the origin of the abnormal proliferating cell clone. In this study, however, cells with a normal c h r o m o s o m e c o m p l e m e n t may represent nonneoplastic cells, such as fibroblasts or normal smooth muscle cells that may have a growth advantage under culture conditions. In the tumors with abnormal karyotypes we report, the vast majority of rearrangements were structural, consequently leading to modal chromosome numbers in the d i p l o i d range. Each chromosome deviation considered specific in l e i o m y o m a s was found in our series, i.e., t(12;14) (3 of 14), del(7) (2 of 14), and trisomy 12 (1 of 14), with the exception of t(1;2) which has been recurrently described by only one laboratory [18]. Furthermore, other rare (M8) and very complex (M9) chromosome changes have been observed with preferential breakpoints at 12q14-15 and 14q23-24 (Fig. 5). Apart from these findings, we wish to emphasize two new, recurrent cytogenetic observations in myomas, i.e., m o n o s o m y 22 and occurrence of ring chromosmnes. We discerned ring chromosomes in two inyomas as the only chromosomal change. One
133
Cytogenetics of 35 Leiomyomas
a
MI2
1
1
b
1 Figure 4 Partial karyotype of case MI2 showing the ring chromosome as the only aberration. Arrows indicate centromeres (a}. C-banding of the ring (b). ring originated from chromosome 1 (M12), and the other was of u n k n o w n origin (M13). Five other myomas with ring chromosomes [two cases with r(1)] have been reported [10, 11, 13, 19]. In two cases, the ring chromosomes were present in addition to t(12;14); the investigators suggest that they are secondary changes [101. In our cases, however, the ring chromosomes were observed as the only change and may well reflect one step of essential importance in leiomyoma development and/or proliferation. Ring chromosomes as sole abnormalities have also been consistently reported in another group of m e s e n c h y m a l tumors. In fat-tissue tumors, rings appear to cause an increased potential for malignant transformation because they are usually observed in lipomas with atypia or in well-differentiated liposarcomas [27, 28]. M12 and M13, however, showed no evidence of elevated n u m b e r s of mitotic figures or atypical nuclear features. Another new observation is the recurrent detection of monosomy 22. In three of our cases (M2, M3, M l l ) , we noted loss of a whole chromosome 22. Two more cases with monosomy 22 [7, 9] in addition to other aberrations and two tumors with at least partial deletion of 22q have been reported [7, 19]. Moreover, loss of chromosome 22 has also been observed as a frequent random change in inyomas with abnormal and normal cell populations [18, 19]. Monosolny 22 or 22q was the first chromosome aberration to be established in a benign tumor, i.e., meningioma [4]. The loss of growth-regulating genes located on 22q may be an important mechanism leading to proliferation of meningeal cells and possibly also of smooth muscle cells. Our statistical study comparing tumors with normal or abnormal karyotypes with the clinicopathologic features (menopausal status and tumor size) showed a random distribution. There was no difference in the clinical and biologic behavior of chromosomally abnormal and normal myomas, which may indicate that karyotypically normal tumors may have alterations at "myoma hot spot regions" at a submicroscopic level.
c_J
{51
T O
~.I~1.~ T ©
6
c,n
d
~" ~ 1 1 I ~ \ 1
o
I
~llllllq~
'
135
Cytogenetics of 35 Leiomyomas
Table 5
Recurrent findings in 84 leiomyomas studied to date (84 abnormal versus 146 normal cases) NO. of cases
Aberration del(7)(q21)/(q21q32) t(12;14)(q14-15;q23-241 Trisomy 12 t(1;2)(p36;p24) Ring chromosomes Monosoiny 22"
Total 16 16 10 5 7 5
Sole abnormality
References
7/16
[5, 7, 9, 10, 17, present study] [7, 9, 15, 17-20, present study] [11. 13-15, 18] [9, 18] [10, 11. 13, 19, present slndy] [7, 9, present study]
10/16 6/10 5/5 2/7
" Also obserw~d as a frequ(.'nt deviation in wlrianl (:ells 19. 18. 1.qJ.
Our results, together with those in previously reported cases, showing recurrent, n o n r an d o m c h r o m o s o m e aberrations, indicate that specific regions of the h u m an genome are preferentially affected in leiomyomas. These genetic changes are generally assumed to be of essential importance for origination and proliferation of leiomynnms. Several critical alterations and breakpoints have been discerned, especially t(12;14)(q14 15;q23-24) and del(7)(q21 22q32), possibly indicating regions where proliferation and transformation associated genes are located. Accordingly, several events may be necessary to induce neoplastic d e v e l o p m e n t and dysregulation of growth in smooth muscle (:ells. This suggestion is supported by the occurrence of several primary aberrations in one tumor and the general concept that neoplastic transformation is a multistep development. Similar cytogenetic observations in myomas and other benign tumors (lipnmas, p l eo m o r p h i c adenomas of the salivary gland, meningiomas), e,g., the frequent occurren(:e of normal karyotypes in such tumors or the finding of several different "subgroups," may indicate that in these tumors benign neoplastic transformation follows similar mechanisms. Moreover, consistent alterations of the same bands (12q in myomas, lipomas, and p l e o m o r p h i c adenomas) and recurrent numerical changes {monosomy 22 in myomas and meningiomas) may indicate that the same growthcontrolling genes can be affected in a variety of benign neoplasms. The signifi(:ance of these recurrently altered (:hromosome regions in myomas must be delineated further by molecular studies, especially of tumors showing a normal (:hromosome complement. This work was supported in part by Grant No. CA-41183 [tom the, National Cancer Institute. l)r. Marion Kiechle-Schwarz is a fellow of the Deutsche Forschungsgemeinschaft (AZ Ki 352-1). The authors thank Fred Flohrschutz and Kelly Schutz for photographic assistance.
REFERENCES 1. Sandberg AA (19801: The Chromosomes in Human Cancer and Leukemia. Elsevier North Holland, New York. pp. 459 462. 2. Sreekantaiah C, Leong SPL, Karakousis CP, McGee DL, Rappaport WD, Villar HV, Nea113, Fleming S, Wankel A, Herrington PN, Carmona R, Sandberg AA (19901: Cytogenetic profile of 109 lipomas. Cancer Res 51:422-433, 3. Mark J, Dahlenfors R (19861: Cytogenetical observations in 1(10 human benign pleomorphic adenomas: Specificity of the chromosomal aberrations and their relationship to sites of localized oncogenes. Anticancer Res 6:299-308. 4. Zang DK (1982): Cytological and cytogenetical studies on human meningoma. Cancer Genet Cytogenet 6:249 274.
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5. Heim S, Nilbert M, Vanni R, Floderus U-M, Mandahl N, Liedgren S, Lecca U, Mitehnan F (1988): A specific translocation, t(12;14)(q14 15;q23-24) characterizes a subgroup of uterine leiomyomas. Cancer Genet Cytogenet 32:13-17. 6. Gibas Z. Griffin CA, Emanuel BS {1988]: Clonal chromosome re.arrangemeuts in a uterine myoma. Cancer Genet Cytogenet 32:19-24. 7. Turc-Carel C, Dal Cin P, Boghosiau L, Terk-Zakarian J, Sandberg AA (1988): Consistent breakpoints in region 14q22 24 in uterine leiomyoma. Cancer Genet Cytogenet 32:25-31. 8. Vanni R, Lecca U (1988): lnw)lvement of the long arm of chromosome 12 in chronmsome rearrangements of uterine leiomyoma. Cancer Genet Cytogenet 32:33-34. 9. Mark ], Havel G, Grepp C, Dahlenfors R, Wedell B [1988): Cytogenetical obserwttions in human benign uterine leiomyomas. Anticancer Res 8:621-626. 10. Nilbert M, Heim S. Mandahl N, Floderus U-M, Willen H, Akerman M, Mitehnan F (1988): Ring formation and structural rearrangements of chromosome 1 as secondary changes in uterine leiomyomas with t(12;14)(q14-15;q23 24). Cancer Genet Cytogenet 36:183-190. 11. Nilbert M, Helm S, Mandabl N, Floderus U-M, Willen H, Mitehnan F (1988): Karyotypic rearrangements in 20 uterine leiomyomas. Cytogenet (;ell Genet 49:300 304. 12. Boghosian L, Dal Cin P, Sandberg AA (1988): An interstitial deletion of chromosome 7 may characterize a subgroup of uterine leiomyoma. Cancer Genet Cytogenet 34:207-208. 13. Vanni R, Nieddu M, Paoli R, Lecca IJ (1989): Uterine [eiomyoma cytogenetics. 1. Rearrangements of chromosome 12. Cancer Genet Cytogenet 37:49-54. 14. Nilbert M, Helm S, Mandahl N, Floderus U-M, Willen H, Mite.lman F (1990): Trisomy 12 in uterine leiomyomas--a new cytogenetic subgroup. Cancer Genet Cytogenet 45:63-66. 15. Havel G, Dahlenfors R, Wedell B, Mark J (1989): Similar cbromosomal evolution in a uterine stromosarcoma and in one of two leiomyomas from the same patient. Acta Pathol Microbinl hnmunol Scand 97:143-146. 16. Mugneret F, Lizard-Nacol S, Volk C, Cuisenier ], Colin F, Turc-Carel C {1988): Association of breakpoint 14q23 with uterine leiomyoma. Cancer Genet Cytogenel 34:201 206. 17. Sait SNJ, Dal Cin P, Ovanessoff S, Sandberg AA (1989): A uterine leiomyoma showing both t(12;14) and de[(7] abnormalities. Cancer Genet Cylogenet 37:157 161. 18. Mark ], Havel G, Grepp C, Dahlenfors R, Wedell B (1990): Chromosomal patterns in human benign uterine leiomyomas. Cancer Genet Cytogenel 44:1-13. 19. Fan S-X, Sreekantaiah C, Berger C, Medchill M, Pedron S, Sandberg AA (1990): Cytogenetic findings in nine leiomyomas of the uterus. Cancer Genet Cytogenet 47:179 190. 20. Nilbert M, Heim S, Mandahl N, Floderus U-M, Willen H, Mitelman F (1989]: Different karyotypic abnormalities, t(1 ;6) and del[7), in two uterine leiomyomas from the same patient. Cancer Genet Cytogenet 42:51-53. 21. Kiechle-Schwarz M, Berger CS, Surti U, Sandberg AA {1990]: Rearrangement of band 10q22 in leiomyoma and leiomyosarcoma of the uterus. Cancer Genet Cytogenet 47:95-100. 22. Sreekantaiah C, Leong SPL, Sandberg AA {1990]: Complex cytogenetic changes in benign neoplasms: Report of six lipomas. Cancer Gene,t Cytogenet 47:113-130. 23. Dal Cin P, Sandberg AA (1989): Chromosome changes in soft tissue tumors: benign and malignant. Cancer Invest 7:63-76. 24. Seabright M (1971): Rapid banding technique for human chromosomes. Lancet 2:971 972. 25. 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). 26. Sachs L (1982): Applied Statistics. A Handbook of Techniques, 5th ed. Springer, New York. 27. Heim S, Mandahl N, Kristoffersson U, Mitelman F, R6ser B, Rydholm A, Willen H (1987): Marker ring chromosome a new cytogenetic subgroup of adipose tissue tumors. Cancer Genet Cytogenet 24:319-326. 28. Turc-Carel C, Dal Cin P, Limon J, Gibas Z, Kakati S, Pietrzak E, Parry D, Li F, Sandberg AA (1987): Cytogenetic and molecular studies of adipose tissue tumors. Cancer Genet Cytogenet 28:33. 29. Nilbert M, Heim S (1990): Uterine leiomyoma cytogenetics. Genes Chromosomes Cancer 2:3-13.
ABSTRACT: Cytagenetic analysis of short-term cultures fl'om 35 leiomyomas of the female genitourinary tract showed abnormal karyotypes in 14 cases. In 11 o~ 14 aberrant tumors, normal cells were also observed. Structural changes were most frequent, resulting in modal chromosome numbers in the diploid range. Our data confirm preferential breakpoint (:lusters at 7q, 12q14-15, and 14q23 24, mainly resulting from consistent, specific chromosome rearrangements such as tI t2.14)[q14 15;q23-24) and del(7)(q21) or del(7)(q22q32). Tagether with previausly published cases, we describe trisamy 12, ring chromosomes, and manosomy 22 as new additianal recurrenl findings in myomas. Statistical analyses of possible caherencies between tumor karyotype (abnormal versus normal) and clinicopathologic data, as well as age of tbe patie.nts, menopausal status, and tumor size showed no correlatians.
INTRODUCTION T h e l o n g - m a i n t a i n e d g e n e r a l c o n c e p t [1] t h a t a n a b n o r m a l c h r o n l o s o n l e p a t t e r n is r e l a t e d to a m a l i g n a n t or p r e m a l i g n a n t state a n d a n o r m a l d i p l o i d k a r y o t y p e to b e n i g n s t a t e s m u s t be r e v i s e d . A n i n c r e a s i n g n u m b e r of r e c u r r e n t c l o n a l c h r o m o s o m e c h a n g e s h a v e b e e n d e t e c t e d i n b e n i g n t u m o r s , s u c h as l i p o m a s [2], p l e o m o r p h i c a d e n o m a s [3], a n d m e n i n g i o m a s [4]. R e c e n t l y , l e i o m y o m a s w e r e a d d e d to t h i s list [ 5 - 2 1 ] . M o r e over, t h e c h r o m o s o m e d e v i a t i o n s f o u n d in s o m e l i p o m a s a n d m y o n l a s w e r e as c o m p l e x as t h o s e u s u a l l y s e e n in a d v a n c e d c a n c e r s [19, 22]. P a r a l l e l c y t o g e n e t i c s t u d i e s of b e n i g n a n d m a l i g n a n t m e s e n c h y m a l n e o p l a s m s o r i g i n a t i n g f r o m fat a n d s m o o t h m u s c l e t i s s u e s h a v e s h o w n a l t e r a t i o n s in t h e s a m e c h r o m o s o m e b a n d s [231. T h u s , c y t o g e n e t i c i n v e s t i g a t i o n s of b e n i g n n e o p l a s m s are e x p e c t e d to e l u c i d a t e o u r u n d e r s t a n d i n g of d i f f e r e n c e s b e t w e e n b e n i g n a n d m a l i g n a n t p r o l i f e r a t i o n a n d m i g h t p r o v i d e i n t e r e s t i n g i n f o r m a t i o n a b o u t t h e role of c e r t a i n c h r o m o s o m e c h a n g e s in t h e e v o l u t i o n of m a l i g n a n t p h e n o t y p e s . C y t o g e n e t i c s t u d i e s of u t e r i n e l e i o m y o m a s h a v e s h o w n r e c u r r e n t , c l o n a l c h r o m o From tile Cancer Center of the Southwest Biomedical Research Institute of Genetrix, Scottsdale, Arizona (M. K.-S., C. S., C. S. B., A, A. S.) Department of Gynecology and Obstetrics, The University Hospital, Freiburg, F. R. G. (M. K.-S.], the Maricopa Medical Center Phoenix, Arizona (S. P., M. T. M.), and tbe Magee Womens Hospital Department of Pathology, Pittsburgh. Pennsylvania (tl. S.). Address reprint requests to: A. A. Sandberg, M.D., The Cancer Center of the Southwest Biomedi(:al Institute af Genetrix, Scottsdale, AZ 85251. Received June 12, 1990; accepted September 18, 1990.
125 © 1991 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet 53:125 136 (1991) 0165-4608/91/$03.50
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M. Kiecble-Schwarz et al. somal alterations, affecting particularly chromosomes 7, 12, and 14 15-9, 11 14, 16 18, 29]: some of these alterations had been suggested to be specific rearrangements. We report detailed cytogenetic observations of 35 myomas from 33 patients and five specimens of adjacent normal myometrium. We discuss our findings and their significan(:e with regard to previously published cytogenetic studies on leiomyomas and other benign neoplasms. We also present possible correlations between clinicopathologic parameters (age, menopausal status, and tumor size) and cytogenetic findings.
MATERIALS A N D METHODS
We cytogenetica]ly analyzed short-term cultures of fresh samples of uterine tumors and c o r r e s p o n d i n g normal m y o m e t r i u m obtained at surgery. All tumors were macroscopically typical leiomyomas. Histologically, they were all benign, with no atypical cells showing nuclear p l e o m o r p h i s m or increased mitotic activity. Some pertinent clinicopathological data of the patients are summarized in Table 1. Tissue cultures and chromosome preparations were made as previously described [21]; this involved disaggregation of the tumors overnight in collagenase (200 U/lnl) and culture in flasks and on cnverslips for a period of 4 - 2 2 days. Harvest time was i n d i v i d u a l i z e d for each flask and coverslip and included Coh:emid exposure, hypotonic shock, and fixation. Metaphases were G-banded using trypsin-(;iemsa [24]. Karyotypes were constructed ac(:ording to the International System for Human Cytogenetic Nomenclature [25]. Statistical significance was calculated by Wih:oxon-MannWhitney II test, as described by Sachs 126]. RESULTS
Thirty-five myoinas from 33 patients and five specimens of normal myometriunl were studied cytogenetically. The detailed cytogenetic and tissue culture data are shown in Tables 2-4. At least five and up to 30 metaphases were analyzed for each sample. Chromosome analysis of cells cultured from adjacent normal myometrial tissue showed a normal karyotype in three cases (N5, N10, N l l ) . Interestingly, in case N14 two cells with trisomy 12 as the only abnormality were found in addition to normal cells. Case N15 failed to grow in culture (Table 2). Eighteen of 35 tumors showed an a p p a r e n t l y normal female karyotype whereas in 14 cases an abnormal chromosome c o m p l e m e n t was found. Three tumors failed to grow in culture. Table 3 shows the detailed cytogenetic data of 18 leiomyomas I c a s e s M 1 5 M32) with a predoiuinantly normal karyotype. Case M30 had a relatively high number of tetraploid cells {20%). Two myomas (M2 and M21) had one cell each with a terminal deletion affecting the long arln of cbrolnosome 7 and the short arm of chromosome 1, respectively, However, with regard to cytogenetic findings in myomas discussed below, the breakpoint 7q22-q23 is of interest. Table 4 shows the detailed cytogenetic results of 14 tumors with abnormal karyotypes. Most of them (M1, M2, M7 Mg, M l l M14) showed cells with a normal karyotype in a d d i t i o n to the abnormal cell line. Three lnyomas (M3, M4, M5) contained only abnormal cells. Both case M6 and case M10 had two abnormal cell clones. The various abnormalities (except M13 and the previously published case M14) are shown in Figures 1 4 b y partial karyotypes. The modal c h r o m o s o m e munber of all deviant cell lines was in the diploid range. Structural rearrangements included deletions, transhications, insertions, and ring c h r o m o s o m e s and occurred in a significantly higher frequency than numerical changes. Loss or gain of whole chromosomes was nbserved in four cases (M2, M3, M6, M l l ) : Case M6 showed trisomy 12 as the only numerical change. Cases M2, M3,
12 7
Cytogenetics of 35 Leiomyomas Table 1
Pertinent clinical data of patients with m y o m a s
Case
Age
1 2 3 4 5" 6 7 8 9 1if' 11 ~ 12 13 14 ~'J' 15 ~' 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3t) 31 32 33 34
35 39 46 46 32 63 34 48 29 39 49 33 32 56 34 45 41 42 51 51 54 42
Pre Pre [)re Pre Pre Post Pre Pre Pre Pre Pre Pre Pre Post Pre Pre Pre .Pre . .
33 37 43 59 46 43 43 4:] 44 61 41 45
Pre Pre Pre Post Pre Pre Pre Pre
35
Menopause
T u m o r size (cm)
Karyotype
10.2 2.0 6.0 20.2 7.5 3.O 4.0 5.0 6.5 3.0 4.(I
Abnormal/normal Abnormal/normal All abnormal All abnormal All abnormal Abnormal/normal Abnormal/normal Abnormal/normal Abnormal/normal Abnormal/normal Abnormal/normal Abnormal/normal Abnormal/normal Abnormal/normal All normal All normal All normal All normal All normal All normal All normal All normal All normal All normal All normal All normal All normal All normal All normal All normal All normal All normal No results No results No results
12.0 11.0 9.5 7.0 7.0 8.0 2.5 .
.
. 3.8 7.5
Post Pre Pre
3.0 11.0 3.0 3.0 1.8 8.5 5.5 10.5 -13.0 6.0
Cases 12 and 24 are the same patient, as are cases 30 and 31. " Corresponding normal myomelrium is analyzed: see Table 2. ~'Previously published.
a n d M l l s h o w e d m o n o s o m y 22 b e s i d e s o t h e r s t r u c t u r a l a b n o r m a l i t i e s : M11 a l s o s h o w e d m o n o s o m y 6. S t r u c t u r a l r e a r r a n g e m e n t s i n v o l v e d d e l e t i o n s a n d r e s u l t e d ill p a r t i a l l o s s of c h r o m o s o m e s e g m e n t s in e i g h t m y o m a s (M2, M 6 - 1 1 , M14). M o s t f r e q u e n t l y , p a r t s of 7q w e r e l o s t (M6, M 7 , M 1 0 ) , a n d it w a s t h e o n l y r e a r r a n g e m e n t in t w o t u m o r s . S e g m e n t s of 14q, 6q, 20q, 2p, a n d 10q w e r e d e l e t e d in o n e c a s e e a c h . T r a n s l o c a t i o n s t h a t m o s t c o m m o n l y i n v o l v e d c h r o m o s o m e s 12 a n d 14 w e r e p r e s e n t i n t h r e e t u m o r s (M1, M2, M3) as a r e c i p r o c a l e v e n t a n d i n d e p e n d e n t l y in c a s e s M4, M5, a n d M10. Ring c h r o m o s o m e s d e r i v e d f r o m c h r o m o s o m e 1 (M12) a n d u n d e f i n a b l e c h r o m o s o m e m a t e rial (M13) w e r e o b s e r v e d in t w o c a s e s . W i t h r e g a r d to i n v o l v e i n e n t of c h r o m o s o m e r e g i o n s a f f e c t e d by s t r u c t u r a l a b e r r a t i o n s in t h e 14 a b n o r m a l m y o m a s , t h e f o l l o w i n g o b s e r v a t i o n s w e r e m a d e : M o s t fre-
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Table 2
C h r o m o s o m a l f i n d i n g s in five s p e c i m e n s of n o r m a l m y o m e t r i u m
Case
Culture time (days)
No. of cells analyzed
Karyotype
N5 N10 N11 N14"
10 8 9-10 9-10
16 15 15 18 2
46,XX 46,XX 46,XX 46,XX 47,XX, + 12
N15
No growth
Abbreviations: N. normal myometrium. " Previously published.
q u e n t l y , b r e a k s o c c u r r e d at 1 2 q 1 4 - 1 5 (five cases), 1 4 q 2 3 - 2 4 (four cases), l p 3 6 (three cases), 6p21 (two cases), a n d 7 q 2 1 . 2 - 2 2 (two cases). F i g u r e 5 s h o w s t h e b r e a k p o i n t d i s t r i b u t i o n (37 b r e a k p o i n t s in all). M u l t i p l e m y o m a s w e r e a n a l y z e d f r o m t w o p a t i e n t s . T u m o r s M 3 0 a n d M31, d e r i v e d from one patient, both showed a normal female chromosome constitution, but M24 a n d M 1 2 , also f r o m o n e p a t i e n t , h a d d i f f e r e n t k a r y o t y p e s (M24:46,XX; M 1 3 : 4 6 , X X / 47,XX, + r). C o m p a r i s o n of t h e c y t o g e n e t i c a l l y a b n o r m a l m y o m a s v e r s u s t u n m r s w i t h a n o r m a l c h r o m o s o m e c o m p l e m e n t w i t h r e g a r d to c l i n i c a l f e a t u r e s (Table 1) s h o w e d n o c l i n i c o c y t o g e n e t i c c o r r e l a t i o n s . No c o r r e l a t i o n b e t w e e n n o r m a l or a b n o r m a l k a r y o t y p e a n d age or m e n o p a u s a l s t a t u s of t h e p a t i e n t s w a s o b s e r v e d . T h e r e a p p e a r e d to be a p o s i t i v e c o r r e l a t i o n b e t w e e n t u m o r size a n d a b n o r m a l k a r y o t y p e : h o w e v e r , n o s t a t i s t i c a l sig-
Table 3
D e t a i l e d c y t o g e n e t i c d a t a o n 18 l e i o m y o m a s w i t h a n a p p a r e n t l y normal female karyotype
Case
Culture time (days)
No. of cells analyzed
Karyotype
M15 M16 M17 M18 M19 M20 M21 M22 M23 M24 M25 M26 M27 M28 M29 M30 M31 M32
10-11 13 4-5 6 4 6 20-22 4-10 7-11 4-10 5 8-13 5-7 4 6-9 7-12 8-9 8-15 8-9
2(I 6 15 20 2(I/1 17 13/1 19/1 2(I 22 13 21 21 2(I 21/2 20/5 18 20
46,XX 46,XX 46,XX 46,XX 46,XX/92,XXXX (lqh +) 46,XX 46,XX/46,XX,del(7)(q22q23) 46.XX/46,XX,deI[1)(p13) 46,XX 46,XX 46,XX 46,XX 46,XX 46,XX 46,XX/92,XXXX 46,XX/92.XXXX 46,XX 46,XX
Abbreviation: M, leiomyoma.
129
C y t o g e n e t i c s of 35 L e i o m y o m a s
Table 4
D e t a i l e d c h r o m o s o m e r e a r r a n g e m e n t s in 14 l e i o m y o m a s of t h e f e m a l e g e n i t o u r i n a r y tract
Case
Culture time (days)
M1
3-5
M2
4
M3 M4
2-7 5
M5 M6
7-8 5-6
M7
5
M8
5-9
M9
5-8
M10
5
Mll
10 18
M12
6
M13
5-7
M14
4-5
Karyotype 46,XX,t(12;14)(q 15:q24) 20 46,XX - 2 45,XX,t(12;14)(q14:q23-24], 22 9 46,XX 6 45,XX,ins[inv(2)(qllq37);15](ql 1 :q12q26),t(12;14](q15:q24), - 22 19 46,XX,t(9;12)(p22;q15) 21 92,XXXX,t(9;12)(p22 ;q15),t(9;12)(p22:q15) 2 46,XX,t(2;12)(q36:q15) 21 46,XX = 7 47,XX,+ 12 5 46,XX,del(7)(q22q35] 4 6 cells with additional nonclonal changes 46,XX = 9 46,XX,del(7)(q21.2q31.2) - 2 46,XX 14 46,XX,del(6)(q23q25) 6 45,XX,del(1)(p34.1p36.1), - 3, + der(3)t(3:?)(p25;?), 6, + der(6)t(6;?) (p21.1;?),del(20)(q11.2q13.3),- 21 = 11 46,XX 9 (one cell: 46,XX,lp ) 46,XX 11 46,XX,t(6;14)(p21.3;q24) 4 45,XX, - 7, - 8, ÷ der(8)t(7;8)(p12;p23) 3 3 cells with additional nonclonal changes 46,XX 13 43,XX, - 1, + der(1)t(1:13)(p36.2 or p36.3;q12),del(2)(p21), 6 , - 1 3 , - 2 2 , - 6 3 cells with additional nonclonal changes 46,XX,r(1?) 11 46,XX = 9 46,XX = 27 47,XX,+r 3 46,XX,del(10)(q22q24) 12 46,XX - 8
n i f i c a n c e w a s o b t a i n e d w i t h W i l c o x o n - M a n n - W h i t n e y U test [26] w i t h regard to t u m o r size a n d k a r y o t y p e ( p v a l u e - 0.4).
DISCUSSION To date, 230 l e i o m y o m a s , i n c l u d i n g t h e c a s e s w e r e p o r t , h a v e b e e n s u c c e s s f u l l y karyotyped. Apparently normal female chromosome constitutions have been reported for 146 t u m o r s in c o n t r a s t to 84 c a s e s (36%) w i t h c l o n a l c h r o m o s o m e a b e r r a t i o n s [ 5 - 2 1 , 29]. In m o s t of t h e a b e r r a n t c a s e s , t h e m o d a l c h r o m o s o m e n u m b e r has b e e n in t h e d i p l o i d range. T h u s , s t r u c t u r a l c h r o m o s o m e c h a n g e s u n e q u i v o c a l l y r e p r e s e n t e d t h e m a j o r i t y of t h e o b s e r v e d d e v i a t i o n s . A c c o r d i n g to t h e a b o v e r e p o r t s a n d o u r o w n i n v e s t i g a t i o n s , c h r o m o s o m e breaks o c c u r in a n o n r a n d o m p a t t e r n a n d c l u s t e r to c h r o m o s o m e r e g i o n s 7 q 2 1 - 3 2 , 1 2 q 1 3 - 1 5 , a n d 1 4 q 2 1 - 2 4 (Fig. 5). A d d i t i o n a l c h r o m o s o m e b a n d s p r e f e r e n t i a l l y rea r r a n g e d are l p 3 6 , 2 p 2 4 - 2 5 , a n d 6p21. T h i s b r e a k p o i n t d i s t r i b u t i o n r e s u l t s
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M. Kiechle-Schwarz et al.
MI 12
14
12
14
rd2 22
M3
i, 7 12
14
M4
15
Ir~*. 9
12
;[
P
22
-,el-
M5
B
12
2
Figure 1
Partial karyolyp(~s of (:as(~s MI to MS.
mainly from recurrently described rearraugements, namely t(12;14) (q13-15;q21-24),del{7)(q21) or del(7)(q21q32), and t(l:2)(p36;p24), shown in detail in Table 5. Deletion 7q and the 12;14 translocation are tbe most consistent findings in myomas and have been reported in 16 cases each. Their frequent occurrence even as sole abnormalities suggests that they are primary specific events playing an important role in myoma development and proliferation: these changes have been the basis for delineation of cytogenetic subgroups of myomas [12, 15]. However, several cases of leiomyomas showing two or more of these recurrent changes at the same time have been reported [17, and present study]. Although numerical changes are relatively rare in myomas, trisomy 12 has been
131
Cytogenetics of 35 Leiomyomas
a
b
M6
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12
7
M7
7
M8
6 Figure 2
Partial karyotypes of cases M6, M7, and MS.
observed in 10 cases (Table 5). Because it was the sole abnormality in six cases, it is considered to constitute another primary specific aberration. In addition to the primary recurrent abnormalities, various structural and nnnmrical changes have been observed in some cases [101, reflecting clonal evolution within the tumor cell populations. Furthermore, some leiomyomas have nonrecurrent chmal abnormalities [11, 18, and present studyl, some showing very complex chromosome rearrangements [19] and affecting the same bands as a leiomyosarcoma 121]. In the present study of 35 leiomyomas, the frequency of normal female chromosome complement was 51% (18 of 35). Abnormal karyotypes were found in 40% {14 of 35), and the failure rate was < 10% [3 of 35). Similar results were obtained by other groups of investigators studying fewer cases, however [9, 18]. In our series, tumors with cytogenetically abnormal and normal cells (11 of 14), more than one abuormal clone (1 of 14), and only one abnormal cell line (3 of 14) were found. The relatively high n u m b e r of apparently normal cells coexisting with abnormal clones appears to represent a c o m m o n feature of benign tuinors, such as meningiomas [4I, pleomorphic
132
M. Kiechle-Schwarz et al.
= 1
6
i 3
6
14
20
21
8
7
6
22
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Figure 3 Partial karyotypes of the myomas with complex chromosome rearrangements (Mg, M10, Mll).
adenomas [3], lipomas [2], and leiomyomas [9, 11, 18]. For these tumors, the normal cell lines, either found alone or in combination with an abnormal clone, have been suggested to represent proliferating neoplastic cells. Detailed studies on tumors with normal stemlines showed that their cells possess a higher chromosome instability as c o m p a r e d with n o n n e o p l a s t i c cell populations, which resulted in formation of variant cells carrying a variety of nonclonal numerical and/or structural aberrations [9, 18]. Certain aberrations may lead to an increasing growth potential and may constitute the origin of the abnormal proliferating cell clone. In this study, however, cells with a normal c h r o m o s o m e c o m p l e m e n t may represent nonneoplastic cells, such as fibroblasts or normal smooth muscle cells that may have a growth advantage under culture conditions. In the tumors with abnormal karyotypes we report, the vast majority of rearrangements were structural, consequently leading to modal chromosome numbers in the d i p l o i d range. Each chromosome deviation considered specific in l e i o m y o m a s was found in our series, i.e., t(12;14) (3 of 14), del(7) (2 of 14), and trisomy 12 (1 of 14), with the exception of t(1;2) which has been recurrently described by only one laboratory [18]. Furthermore, other rare (M8) and very complex (M9) chromosome changes have been observed with preferential breakpoints at 12q14-15 and 14q23-24 (Fig. 5). Apart from these findings, we wish to emphasize two new, recurrent cytogenetic observations in myomas, i.e., m o n o s o m y 22 and occurrence of ring chromosmnes. We discerned ring chromosomes in two inyomas as the only chromosomal change. One
133
Cytogenetics of 35 Leiomyomas
a
MI2
1
1
b
1 Figure 4 Partial karyotype of case MI2 showing the ring chromosome as the only aberration. Arrows indicate centromeres (a}. C-banding of the ring (b). ring originated from chromosome 1 (M12), and the other was of u n k n o w n origin (M13). Five other myomas with ring chromosomes [two cases with r(1)] have been reported [10, 11, 13, 19]. In two cases, the ring chromosomes were present in addition to t(12;14); the investigators suggest that they are secondary changes [101. In our cases, however, the ring chromosomes were observed as the only change and may well reflect one step of essential importance in leiomyoma development and/or proliferation. Ring chromosomes as sole abnormalities have also been consistently reported in another group of m e s e n c h y m a l tumors. In fat-tissue tumors, rings appear to cause an increased potential for malignant transformation because they are usually observed in lipomas with atypia or in well-differentiated liposarcomas [27, 28]. M12 and M13, however, showed no evidence of elevated n u m b e r s of mitotic figures or atypical nuclear features. Another new observation is the recurrent detection of monosomy 22. In three of our cases (M2, M3, M l l ) , we noted loss of a whole chromosome 22. Two more cases with monosomy 22 [7, 9] in addition to other aberrations and two tumors with at least partial deletion of 22q have been reported [7, 19]. Moreover, loss of chromosome 22 has also been observed as a frequent random change in inyomas with abnormal and normal cell populations [18, 19]. Monosolny 22 or 22q was the first chromosome aberration to be established in a benign tumor, i.e., meningioma [4]. The loss of growth-regulating genes located on 22q may be an important mechanism leading to proliferation of meningeal cells and possibly also of smooth muscle cells. Our statistical study comparing tumors with normal or abnormal karyotypes with the clinicopathologic features (menopausal status and tumor size) showed a random distribution. There was no difference in the clinical and biologic behavior of chromosomally abnormal and normal myomas, which may indicate that karyotypically normal tumors may have alterations at "myoma hot spot regions" at a submicroscopic level.
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135
Cytogenetics of 35 Leiomyomas
Table 5
Recurrent findings in 84 leiomyomas studied to date (84 abnormal versus 146 normal cases) NO. of cases
Aberration del(7)(q21)/(q21q32) t(12;14)(q14-15;q23-241 Trisomy 12 t(1;2)(p36;p24) Ring chromosomes Monosoiny 22"
Total 16 16 10 5 7 5
Sole abnormality
References
7/16
[5, 7, 9, 10, 17, present study] [7, 9, 15, 17-20, present study] [11. 13-15, 18] [9, 18] [10, 11. 13, 19, present slndy] [7, 9, present study]
10/16 6/10 5/5 2/7
" Also obserw~d as a frequ(.'nt deviation in wlrianl (:ells 19. 18. 1.qJ.
Our results, together with those in previously reported cases, showing recurrent, n o n r an d o m c h r o m o s o m e aberrations, indicate that specific regions of the h u m an genome are preferentially affected in leiomyomas. These genetic changes are generally assumed to be of essential importance for origination and proliferation of leiomynnms. Several critical alterations and breakpoints have been discerned, especially t(12;14)(q14 15;q23-24) and del(7)(q21 22q32), possibly indicating regions where proliferation and transformation associated genes are located. Accordingly, several events may be necessary to induce neoplastic d e v e l o p m e n t and dysregulation of growth in smooth muscle (:ells. This suggestion is supported by the occurrence of several primary aberrations in one tumor and the general concept that neoplastic transformation is a multistep development. Similar cytogenetic observations in myomas and other benign tumors (lipnmas, p l eo m o r p h i c adenomas of the salivary gland, meningiomas), e,g., the frequent occurren(:e of normal karyotypes in such tumors or the finding of several different "subgroups," may indicate that in these tumors benign neoplastic transformation follows similar mechanisms. Moreover, consistent alterations of the same bands (12q in myomas, lipomas, and p l e o m o r p h i c adenomas) and recurrent numerical changes {monosomy 22 in myomas and meningiomas) may indicate that the same growthcontrolling genes can be affected in a variety of benign neoplasms. The signifi(:ance of these recurrently altered (:hromosome regions in myomas must be delineated further by molecular studies, especially of tumors showing a normal (:hromosome complement. This work was supported in part by Grant No. CA-41183 [tom the, National Cancer Institute. l)r. Marion Kiechle-Schwarz is a fellow of the Deutsche Forschungsgemeinschaft (AZ Ki 352-1). The authors thank Fred Flohrschutz and Kelly Schutz for photographic assistance.
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5. Heim S, Nilbert M, Vanni R, Floderus U-M, Mandahl N, Liedgren S, Lecca U, Mitehnan F (1988): A specific translocation, t(12;14)(q14 15;q23-24) characterizes a subgroup of uterine leiomyomas. Cancer Genet Cytogenet 32:13-17. 6. Gibas Z. Griffin CA, Emanuel BS {1988]: Clonal chromosome re.arrangemeuts in a uterine myoma. Cancer Genet Cytogenet 32:19-24. 7. Turc-Carel C, Dal Cin P, Boghosiau L, Terk-Zakarian J, Sandberg AA (1988): Consistent breakpoints in region 14q22 24 in uterine leiomyoma. Cancer Genet Cytogenet 32:25-31. 8. Vanni R, Lecca U (1988): lnw)lvement of the long arm of chromosome 12 in chronmsome rearrangements of uterine leiomyoma. Cancer Genet Cytogenet 32:33-34. 9. Mark ], Havel G, Grepp C, Dahlenfors R, Wedell B [1988): Cytogenetical obserwttions in human benign uterine leiomyomas. Anticancer Res 8:621-626. 10. Nilbert M, Heim S. Mandahl N, Floderus U-M, Willen H, Akerman M, Mitehnan F (1988): Ring formation and structural rearrangements of chromosome 1 as secondary changes in uterine leiomyomas with t(12;14)(q14-15;q23 24). Cancer Genet Cytogenet 36:183-190. 11. Nilbert M, Helm S, Mandabl N, Floderus U-M, Willen H, Mitehnan F (1988): Karyotypic rearrangements in 20 uterine leiomyomas. Cytogenet (;ell Genet 49:300 304. 12. Boghosian L, Dal Cin P, Sandberg AA (1988): An interstitial deletion of chromosome 7 may characterize a subgroup of uterine leiomyoma. Cancer Genet Cytogenet 34:207-208. 13. Vanni R, Nieddu M, Paoli R, Lecca IJ (1989): Uterine [eiomyoma cytogenetics. 1. Rearrangements of chromosome 12. Cancer Genet Cytogenet 37:49-54. 14. Nilbert M, Helm S, Mandahl N, Floderus U-M, Willen H, Mite.lman F (1990): Trisomy 12 in uterine leiomyomas--a new cytogenetic subgroup. Cancer Genet Cytogenet 45:63-66. 15. Havel G, Dahlenfors R, Wedell B, Mark J (1989): Similar cbromosomal evolution in a uterine stromosarcoma and in one of two leiomyomas from the same patient. Acta Pathol Microbinl hnmunol Scand 97:143-146. 16. Mugneret F, Lizard-Nacol S, Volk C, Cuisenier ], Colin F, Turc-Carel C {1988): Association of breakpoint 14q23 with uterine leiomyoma. Cancer Genet Cytogenel 34:201 206. 17. Sait SNJ, Dal Cin P, Ovanessoff S, Sandberg AA (1989): A uterine leiomyoma showing both t(12;14) and de[(7] abnormalities. Cancer Genet Cylogenet 37:157 161. 18. Mark ], Havel G, Grepp C, Dahlenfors R, Wedell B (1990): Chromosomal patterns in human benign uterine leiomyomas. Cancer Genet Cytogenel 44:1-13. 19. Fan S-X, Sreekantaiah C, Berger C, Medchill M, Pedron S, Sandberg AA (1990): Cytogenetic findings in nine leiomyomas of the uterus. Cancer Genet Cytogenet 47:179 190. 20. Nilbert M, Heim S, Mandahl N, Floderus U-M, Willen H, Mitelman F (1989]: Different karyotypic abnormalities, t(1 ;6) and del[7), in two uterine leiomyomas from the same patient. Cancer Genet Cytogenet 42:51-53. 21. Kiechle-Schwarz M, Berger CS, Surti U, Sandberg AA {1990]: Rearrangement of band 10q22 in leiomyoma and leiomyosarcoma of the uterus. Cancer Genet Cytogenet 47:95-100. 22. Sreekantaiah C, Leong SPL, Sandberg AA {1990]: Complex cytogenetic changes in benign neoplasms: Report of six lipomas. Cancer Gene,t Cytogenet 47:113-130. 23. Dal Cin P, Sandberg AA (1989): Chromosome changes in soft tissue tumors: benign and malignant. Cancer Invest 7:63-76. 24. Seabright M (1971): Rapid banding technique for human chromosomes. Lancet 2:971 972. 25. 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). 26. Sachs L (1982): Applied Statistics. A Handbook of Techniques, 5th ed. Springer, New York. 27. Heim S, Mandahl N, Kristoffersson U, Mitelman F, R6ser B, Rydholm A, Willen H (1987): Marker ring chromosome a new cytogenetic subgroup of adipose tissue tumors. Cancer Genet Cytogenet 24:319-326. 28. Turc-Carel C, Dal Cin P, Limon J, Gibas Z, Kakati S, Pietrzak E, Parry D, Li F, Sandberg AA (1987): Cytogenetic and molecular studies of adipose tissue tumors. Cancer Genet Cytogenet 28:33. 29. Nilbert M, Heim S (1990): Uterine leiomyoma cytogenetics. Genes Chromosomes Cancer 2:3-13.
