Chromosome analysis of 96 uterine leiomyomas

Chromosome Analysis of 96 Uterine Leiomyomas Nikos Pandis, Sverre Heim, Georgia Bardi, Ulla-Maria Flod6rus, Helena Will6n, Nils Mandahl, and Felix Mitelman

ABSTRACT: From September 1989 to May 1990, we attempted cytogenetic analysis on 96 uterine leiomyomas removed from 64 women. Of the 90 tumors in which analysis was successful, 59 had a normal karyotype while 31 had clonal abnormalities. The most common aberration (13 tumors) was 7 q - , mostly del(7)(q21.2q31.2); in two tumors with +12 end t(12;14) as the primary abnormalities, the 7 q - was obviously a secondary change since it was found only in a subclone. A t(12:14)(q14-15;q23-24) was detected in two tumors, complex aberrations involving both 12q14-15 and 14q23-24 were also present in two, and rearrangements of 12q without concomitant 14q changes were seen in another two myomas. Rearrangements of 6p were present in five tumors, and trisomy 12 was found in two. More than one abnormality could be detected in 17 leiomyomas. Evidence of clonal evolution in the form of subclones was found in eight tumors, all of which were cellular and had histologically detectable mitotic activity. In addition to their clonal complexity, these myomas also frequently exhibited clonal telomeric associations (four tumors) and ring chromosome formation (three tumors; twice affecting chromosome 1). Monosomy 22 occurred as a secondary abnormality in three tumors; it, too, may reflect a preferred pathway in the karyotypic evolution of uterine leiomyomas.

INTRODUCTION The first c h r o m o s o m e aberrations in uterine leiomyomas were described 3 years ago, w h e n t(12;14)(q14-15;q23-24) was s h o w n to be a specific karyotypic abnormality [1-3]. Subsequent reports [4-19] have expanded the data base so that now more than 100 myomas with clonal c h r o m o s o m e changes are available for evaluation. The karyotypic picture has become more complex, and at least four cytogenetically abnormal subgroups of uterine leiomyoma, characterized by the presumably primary abnormalities t/del(6p), del(7q), +12, and t(12;14), have emerged [20]. It seems self-evident that our understanding of m y o m a cytogenetics will undergo further refinements w h e n n e w cases are added. We here describe the cytogenetic analysis of another 96 tumors, 31 of w h i c h had clonal c h r o m o s o m e abnormalities.

From the Departments of Clinical Genetics (N. P., S. H., G. B., N. M., F. M.). Gynecology(U.-M. F.), and Clinical Pathology (H. W.), University Hospital, Lund, Sweden. Address reprint requests to: Dr. Sverre Heim, Department of Clinical Genetics, University Hospital, S-221 85 Lund, Sweden. Received September 25, 1990; accepted December 10, 1990.

11 .g' 1991 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010

Cancer Genet Cytogenet 55:11-18 (1991) 0165-4608/91/$03.50

12

N. Pandis et al.

Figure 1 del(7)(q21.2q31.2) (partial karyotype from case 67) is the most common cause of a 7q- in uterine leiomyoma.

MATERIALS AND METHODS Fresh tumor samples from 96 histologically verified uterine leiomyomas from 64 w o m e n were minced with scissors and disaggregated in collagenase. The collagenase concentration varied between 0.2% and 0.8% (235 U/mg and 940 U/mg) and the incubation time between 3 hours and 36 hours. The resulting cell suspensions were washed and plated on glass chamber slides in RPM11640 m e d i u m with HEPES buffer, s u p p l e m e n t e d with fetal calf serum (17%), L-glutamine (0.23 mg/ml), and antibiotics (penicillin 100 IU/ml; streptomycin 0.2 mg/ml). After 3 - 8 days, the cultures were harvested in situ by Colcemid exposure, followed by hypotonic treatment in 0.3% NaC1 and fixation in acetic acid : methanol (1 : 3; gradual replacement of the hypotonic solution by fixative). The chromosomes were banded with Wright stain. The subsequent cytogenetic analysis followed the recommendations of ISCN [21]. To be certain that all clonal aberrations reflected in vivo abnormalities, the additional requirement was made that a rearrangement had to be found in at least two different in situ preparations in order to be accepted as clonal.

RESULTS Of the 96 tumor samples, 90 could be successfully analyzed (infection in 1 case, no cell attachment in 2 cases, insufficient n u m b e r of mitoses in 3 cases). Clonal chromosome abnormalities were found in 31 tumors (Table 1) from 31 women. The most c o m m o n aberration was 7 q - (13 tumors), mostly del(7){q21.2q31.2) (Fig. 1). Rearrangements of 12q14-15 were detected in six tumors; twice as the classical t(12;14)(q14-15;q23-24) (Fig. 2), twice as complex abnormalities involving 12q and 14q (Fig. 3), and in two tumors as 12q rearrangement without concomitant 14q changes. The reverse situation, 14q aberration without 12q involvement, was never seen. Other recurring abnormalities were + 12 (two tumors), - 2 2 (three tumors; always as one of several changes), and r(1) (two tumors; a third tumor had an unidentified ring). Another set of n o n r a n d o m aberrations, of 6p, was found in five cases, but in no two tumors as exactly the same rearrangement. More than one clonal aberration was found in 17 myomas, and subclones were present in 8. This group of tumors with clonal evolution encompassed all cases with ring chromosomes and telomeric associations (Fig. 4).

Figure 2 Partial karyotype (case 61) illustrating the t(12;14)(q15;q24) that is a specific abnormality in leiomyoma. The der(12)t(12;14) is to the extreme left and the der(14)t(12;14) is to the extreme right.

Chromosome

13

A n a l y s i s of 96 U t e r i n e L e i o m y o m a s

Table 1

Thirty-one karyotypically abnormal uterine leiomyomas

Case a no.

Lab no.

9 10 14 18 20 24 26 37 ~ 43 44 ~: 46 47 ':

2453-89 2508-90 2606-89 2642-89 2714-89 2752-89 2786-89 2873-89 2917-89 2931-89 2982-89 2983-89

Karyotype 46,XX,del(7](q21.2q32)/46,XX 46,XX,del(7](q21q32)/46,XX 46,XX,del(7)(q21q31)/46,XX 47,XX, + mar/46,XX 47,XX, + 12 46,XX,del(7)(q21q32)/46,XX 47,XX, + mar/46,XX 46,XX,inv(12)(q15q24.3)/46,XX 46,XX,t(6;12)(p22;q13),del(7)(q2 lq31) 47,XX, + 12/47,XX, + 12,t(4;6)(p15;q24),del(7)(q21q31) 45-46,XX,- 7,del(5)(p13),der(13)t(5;13)(p13;q22), + mar/46,XX 46,XX,r(1) (cen--~p36::p 22--* p36::p22--~p36::q32-*cen)/

Cells per clone 11/89 5/20 10/50 4/46 25 59/1 4/16 5/12 13 24/2 7/68 136/3/2/6/4/46

92,XXXX,r(1),r(1)/46,XX,dup[r(1) ]/46,XX,del( 7)(q21q31)/

48

3032-89

50 58':

3145-89 3348-89

60 61 ~'c

3369-89 0089-90

64 67 71 72 73 75

0310-90 O358-9O 0400-90 0444-90 0445-90 0512-90 0689-90

77 ~'':

46,XX,del(3)(q2 lq36),t(6;?)(pll;?),del(7)(q2 lq31),del(11)(q14), t(21;?)(q22;?)/46,XX 46,XX,t(X;3;1)(q24;q25;p36)/ 46,XX,t(1;11)(q25;p15),del(7)(q2 lq31)/46,XX 47,XX, + mar/46,XX 46,XX, - 1, - 10, + der(1)t(1;8)(q25;p21),der(8)del(8)(p21)inv(8) (qllq24), + der(10)t(1;101(q25;p13)/46,XX,- 1 , - 10, + der(1) t(1;8)(q25;p21),inv(7)(q31q34],der(8)del(8)(p21)inv(8] (qllq24), + der(10)t(1;10)(q25;p13)/46,XX 46,XX,t(5;6)(q31;q27)/46,XX 46,XX,t(12;14)(q15;q24)/45,XX,- 22,t(12;14)(q15;q24)/ 45,XX, - 12, - 14, - 1 4 , - 22, + der(12)t(12;14)tas(12p;14q), + der(14)t(12;14)/45,XX,- 12, - 1 4 , - 1 4 , - 19, - 21, - 22, + der(12)t(12;14)tas(12p;19p), + der(14)t(12;14), + tas(14q;21p)/ 46,XX, - 12, - 14, - 14, - 22, + der(12)t(12;14)tas(12p;14q;14q), + der(14)t(12;14)/46,XX,del(7)(q21q31),t(12;14)/46,XX,- 12, - 1 4 , - 14,del(7)(q21q31), + der(12)t(12;14)tas(12p;14q), + der(14)t(12;14)/46,XX,- 1 2 , - 1 4 , - 1 4 , - 21,de1(7), + der(12)t(12 ;14), + tas(14q;21q)/46,XX,- 1 2 , - 1 4 , - 14,de1(7), + der(12)t(12;14)tas(12p;12p), + der(14)t(12;14)/46,XX 46,XX,ins(6;6)(p21.3;q22q24),inv(8)(p2 lq11)/46,XX 46,XX,del(7)(q21.2q31.2)/46,XX 46,XX,del(7)(q21q31)/46,XX,del(7),tas(3p;9p)/46,XX 46,XX,t(10;11)(p13;q25)/46,XX 46,XX,del(7)(q21.2q31.2]/46,XX 46,XX,del(7)(q2 lq31)/46,XX 4 6 , X X , - 3, - 4, + der(3)t(3;?)(p21;?), + der(4)t(3;4)(p21;p12), t(12;14)(q14-55;q23-24)/ 4 5 , X X , - 1 , - 3, - 4, + der(3), + der(4),t(12;14)/ 4 5 - 4 6 , X X , - 1 , - 3 , - 4, + der(3), + der(4),t(12;14), + r/ 4 6 , X X , - 3 , - 4,r(1)(p36q25), + der(3), + der(4),t(12;14)/ 4 6 , X X , - 3 , - 4,r(1)(p36q42], + der(3), + der(4),t(12;14)/ 46,XX, - 3, - 4,r(1)(p36q42),tas(lq;12q), + der(3), + der(4),t(12;14)/ 46,XX, - 3, - 4,r(1)(p36q44], + der(3), + der(4),t(12;14)/ 4 6 , X X , - 3, - 4,r(1)(p36q44),tas[1q;12q), + der(3), + der(4),t(12;14)/ 4 6 , X X , - 3 , - 4,r(1)(p36--~q42 ::q21--*q42), + der(3), + der(4),t(12;14)/ 46,XX, - 3, - 4,r(1)(p36--~q44::q21-*q42), + der(3), + der(4),t(12;14)/ 46,XX, - 3, - 4,r(1)(p36--*q42 ::q21--~q42::q21--~q42), + der(3), + der(4),t(12;14)/46,XX, - 3, - 4,r(1)(p36-*q42::q21--~q42), r(1)(p36q42), + der(3), + der(4),t(12;14)/46,XX

2/2/31 4/21 15/32/1

12/20 5/10/4/2/5/ 88/6/3/2/25

19/12 22/3 18/3/8 28/2 20/10 12/17 24/11/3/3/ 56/2/ 32/2/24/23/ 17/2/51

(continuedl

14

N. P a n d i s et al.

Table 1

Continued

Case ° no.

Lab no.

80

0877-90

83 84

0920-90 1041-90

88

1189-90

91

1266-90

92 96 ~J

1284-90 1423-90

Cells per clone

Karyotype 4 5 , X X , - 1,del(15)(q15),der(19)t(1;19)(q12;q13)/ 4 4 , X X , - 1 , - 22,del(15),der(19)/ 43,XX, 1, - 14, - 15, - 22,der(19), + mar/46,XX 46,XX,t(2;6/(q37;p21),del(6)(q21q23)/46,XX 4 4 , X X , - 3 , - 4,t{X;?) (q26;?),der(3)t(3;3) (p24;q13),der(5)t(5;?) (q31 ;?), der(6) inv(6) (p2 lp22) t(q22;?), der(12) t(12 ;?) (q13 ;?), der(14)t(14;?)(q22;?)/44,XX,-3,- 4,der(X)t{X;?),der(3)t(3;3), der(5)t(5;?)tas(5p;19q),der(6)inv(6)t(q22;?),der(12)t(12;?),t(14;?)/ 44,XX, - 3, - 4,der(X)t(X;?),der(3)t(3 ;3),der(5)t(5;?), der(6)inv(6)t(q22;?),t(12;?)t(14;?),tas(2q;16p)/44,XX, - 3, - 4, t(X;?),der(3)t(3;3),der(5)t(5;?),der(6)inv(6)t(q22;?), der(12)t(12 ;?),der(14)t(14;?),tas(16p;21p)/44,XX, - 3, - 4, der(X)t(X;?), der(3)t(3;3),der(5)t(5;?),der(6)inv(6)t(q22;?), der(12)t(12;?),der(14)t(14;?),tas(19q;19q)/44,XX, - 3, - 4 , der(X)t(X;?),der(3)t(3;3),der(5)t(5;?),der(6)inv(6)t(q22;?), der(12)t(12;?),t(14;?),tas(19q;21p)/45,XX, - 3, - 4,t(X;?),der(3), der(5),der(6),t(12;?),t(lq;?), +r/45,XX, - 3, 4,t(X;?J,der(3), der(5),der(6),t(12;?),der(14)t(14;?), + mar 43,XX, - 2, - 7, - 11, - 11, - 15, - 22,de1{1)(q42),der(3)t(3;?)(q12,?), del(4) (q31),del(6) (q15),der(8)t(7;8)(p14;p12)t(6;8) (q15;q24), del(9)(p13),der(11)t(2;11)(q11;p15),der(12)t(12;?)(q15;?), + 3mar/ 46,XX 44,X, - X, - 4, - 13,der(1)t(1;2) (p32;p21),der(2)t(2;13)(p21;q12), del(7)(p15)(q2 lq31),der(12/(pter--~q13::?::14q24--, 14qter), der(14)t(14;?)(q24;?),der(16)t(12;16)(q15;q24), +mar/45,XX, 4, 13,der(1),der(2),del(7),der(12),der(14)t(14;?),der(16), + mar/ 46,XX 46,XX,del(5)(q13q15),del(7)(qllq32),del(12){q13q15)/46,XX 46,XX,der(7)t(7;12)(q31.2;q21.2),der(9) inv(9)(p24q11)ins(9;13)(qll;q13q21.2), der(12)t(12;14)(q15;q24.1),del(13)(q13q21.2), der(14)(14pter-*q24.1::12q15-* 12q21.2:),?t(15;21)(p11-13;q21)

32/5/8/10

43/7 23/5/2/3/2/ 7/32/4

5/26

6/20/4

27/1 32

~ Consecutive numbering 1-96. b Previously reported in [16]. Previously reported in [171.

DISCUSSION O u r i n i t i a l e x p e r i e n c e w i t h r e l a t i v e l y s h o r t (3 h o u r s to o v e r n i g h t ) c o l l a g e n a s e t r e a t m e n t l e d u s to c o n c l u d e t h a t t h i s o f t e n r e s u l t e d in i n s u f f i c i e n t d i s a g g r e g a t i o n o f t h e t o u g h m y o m a t i s s u e . W h e n w e e x t e n d e d t h e 0 . 2 % c o l l a g e n a s e t r e a t m e n t to 3 0 - 3 6 h o u r s , m o r e c o m p l e t e d i s a g g r e g a t i o n w a s a c h i e v e d , i n its t u r n l e a d i n g to b e t t e r cell a t t a c h m e n t u p o n p l a t i n g a n d to a n a p p r o x i m a t e d o u b l i n g of t h e f r e q u e n c y of c y t o g e n e t i c a l l y a b n o r m a l c a s e s ( f r o m a b o u t o n e - f o u r t h to o n e - h a l f ; r e s u l t s n o t s h o w n ) . T h e f i n d i n g s i n t h e 31 t u m o r s w i t h c l o n a l c h r o m o s o m e a b n o r m a l i t i e s c o n f i r m t h e

F i g u r e 3 Partial karyotype of the der(12) (12pter-12q13::?::14q24-14qter), der(14)t(14;?)(q24;?), and der(16)t(12;16) (q15;q24) found in case 91. The c h r o m o s o m e s are, from left to right: normal 12, der(12), normal 14, der(14), normal 16, and der(16).

Chromosome Analysis of 96 Uterine Leiomyomas

15

f i~

Figure 4 Ring chromosomes and telomeric associations were present only in cellular myomas showing clonal evolution. Metaphase plate from case 77, illustrating (in addition to other aberrations; see Table 1) a tas(1q;12q) and an r(1).

existence of at least four different cytogenetic subgroups [20]. The most c o m m o n aberration (13 tumors) was an interstitial deletion leading to 7 q - , and often the breakpoints could be m a p p e d at subband level, enabling us to designate the aberration del(7)(q21.2q31.2) (Fig. 1). The consistency with w h i c h exactly these breakpoints are found in the del(7q) of l e i o m y o m a s / r e f e r e n c e s 4, 6~ 12, 34, and 18, in a d d i t i o n to the present series) distinguishes it from the del(7q) of secondary m y e l o d y s p l a s i a and leukemia [23]. It is possible that m y o m a cells have an intrinsic p r o p e n s i t y for breaks at these very sites. Alternatively, the lesson to be learned from the cytogenetic data might be that the pathogenetically essential outcome of the deletion is not just the loss of genetic material from somewhere between 7q21 and 7q31, but instead some kind of position effect exerted on loci in the breakpoint regions. Earlier findings of del(7q) and other myoma-associated abnormalities, in particular t(12;14), coexisting in the same clone have raised uncertainties as to whether del(7q) should be v i e w e d as a primary or secondary aberration [18, 20]. Cases 44 and 61 of this series, in w h i c h del(7q) was found in subclones only, whereas the primary abnormalities trisomy 12 (case 44) and t(12;14) (case 61) were c o m m o n to all clones, prove the secondary nature of del(7q) in at least these tumors.

16

N. Pandis et el. Trisomy 12, another k n o w n leiomyoma-associated aberration [13], was detected in cases 20 and 44. It is worthy of note that whereas mitoses with a normal c h r o m o s o m e c o m p l e m e n t can almost always be found in a d d i t i o n to the cytogenetically abnormal clones in short-term m y o m a cultures, this is not so if the aberration is + 12. Even after as long as 4 weeks, the trisomy 12-containing clone c o m p l e t e l y dominates the culture so that no 46,XX cells are found. Trisomy 12 has in other contexts been associated with chronic l y m p h a t i c leukemia [24-26] and benign ovarian tumors [27, 28]. The classical [1-3] t(12;14)(q14-15;q23-24) (Fig. 2) was found in cases 61 and 77, complex aberrations of 12q and 14q (Fig. 3) coexisted in cases 84 and 91, and 12q rearrangements without simultaneous 14q changes were seen in cases 37 and 43. The findings in the latter two tumors add to the now substantial subgroup of m y o m a s with 12q but no 14q abnormalities [15, 20]. The reverse situation, on the other hand, has been described only once [5]. In our opinion, one should nevertheless refrain from too strenuous predictions about the pathogenetic relationship between t(12;14) and other 12q13-15 changes based on these data. It is possible that even those tumors that have no cytogenetically visible 14q rearrangement may have submicroscopic involvement of the relevant chromosome 14 loci, and that a reciprocal 12;14-rearrangement thus exists in all myomas with visible 12q aberrations. The situation w o u l d then parallel what has been found in variant Ph translocations in chronic m y e l o i d leukemia. As long as the myoma-associated loci on c h r o m o s o m e s 12 and 14 have not been defined in molecular terms, the testing of this hypothesis remains impossible. The exact breakpoints in t(12;14) have been uncertain; the alternatives t(12;14)(q14;q23) and t(12;14)(q15;q24) w o u l d result in very similar derivative chromosomes. Based on the 12q and 14q rearrangements presented here, in particular the results of cases 37, 61, 77, and 91, we are inclined to c o n c l u d e that the breakpoints are in 12q15 and 14q24.1 [16]. If this interpretation is substantiated in future studies, the reciprocal 12;14-rearrangement of leiomyomas should be written t(12:14)(q15;q24.1). A further consequence of the precision m a p p i n g of the leiomyome-associated 12q breakpoint is that it now seems clear that this breakpoint must be distal to the one affected in the t(12;16) of m y x o i d liposarcoma [29, 30]. On the other hand, the 12q abnormalities of lipomas [31] and salivary gland p l e o m o r p h i c a d e n o m a s [32, 33] might still affect the same loci that are arranged in leiomyomas. Although no abnormalities of the short arm of c h r o m o s o m e 6 were recurrent in the present series, various rearrangements of 6p were nevertheless detected in five tumors, confirming the suggestion that der(6p) is a n o n r a n d o m change in l e i o m y o m a s (14). The diversity of the 6p changes and the fact that they are mostly found together with other aberrations, including t(12;14) and + 12, suggest that they reflect secondary events. More than one abnormality was present in 17 myomas and subclones could be identified in 8; both findings are evidence of clonal evolution within the tumor cell population. In general, this cytogenetic progression was paralleled by histologic progression (results not shown), with all eight tumors displaying clonal evolution belonging to the cell-rich l e i o m y o m a subset that also had histologically detectable mitotic activity [17, 22]. Prominent among the secondary aberrations were, in a d d i t i o n to the already mentioned del(7q) found in two tumors, ring chromosomes, twice involving chromosome 1. These findings confirm earlier observations [9] that r(1) formation is a preferred p a t h w a y in the clonal evolution of uterine leiomyomas. Another manifestation of telomeric hyperreactivity, telomeric association, was as a clonal p h e n o m e n o n detected in four tumors. Apart from its occurrence during clonal evolution in myomas, telomeric association seems to be particularly frequent in malignant fibrous histiocytomas [34, 35]. Its pathogenetic significance remains un-

C h r o m o s o m e A n a l y s i s of 96 U t e r i n e L e i o m y o m a s

17

k n o w n . F i n a l l y , loss of o n e c h r o m o s o m e 22, w h i c h is o t h e r w i s e r e g u l a r l y a s s o c i a t e d w i t h m e n i n g i o m a s [36, 37], o c c u r r e d i n t h r e e t u m o r s w i t h a c o m p l e x k a r y o t y p e . M o n o s o m y 22 h a s also p r e v i o u s l y b e e n d e t e c t e d i n m y o m a s [1, 3, 11, 15], a n d w e c o n c l u d e t h a t it too p r o b a b l y r e p r e s e n t s a n o n r a n d o m s e c o n d a r y a b n o r m a l i t y .

This work was supported by grants from the Swedish Cancer Society and the Ingabritt and Arne Lundberg's Research Foundation. Drs. Pandis and Bardi are on leave from the Papanikolaou Research Center, Hellenic Anticancer Institute, Athens, Greece. Their visit to Sweden was made possible by financial support from the Swedish Institute.

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