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Complex karyotypic changes, including rearrangements of 12q13 and 14q24, in two leiomyosarcomas
Complex Karyotypic Changes, Including Rearrangements of 12q13 and 14q24, in Two Leiomyosarcomas Mef Nilbert, Nils Mandahl, Sverre Heim, Anders Rydholm, GOran Helm, Helena Will,n, Bo Baldetorp, and Felix Mitelman
ABSTRACT: Cytogenetic investigation of short-term cultures from two leiomyosarcomas revealed complex karyotypic changes in both cases. The first tumor, a subcutaneous leiomyosarcoma of the knee, had the karyotype 7 0 - 8 0 , X Y , + X , + Y , + l , + l , + 2 , + 2 , + 3 , + 3 , + 4 , + 4 , + 7 , + 7 , + 8 , + 8 , + 9, +10, +15, +15, +16, +16, +18, +19, + 20, + 21, + 21, + 22, + 22,t(?;5)(5;21)(?;q35p11;q11), t(?;5)(5;21)(?;q35pl 1;ql 1), + del(11)(q22),der(13)t(12;13)(q13;q22),der(14)t(9;14)(pl 1;pl 1), + 1 4 p + , + t(20;?)(q13;?), +t(20;?)(q13;?), + 2 mar. A polyploidized clone with 120-150 chromosomes was also abserved. DNA flow cytometry revealed only one abnormal peak, corresponding to a DNA index of 1.76. The other tumor, a uterine leiomyosarcoma, had the karyotype 61-67, X, - X , + 1, + 3, + 5, + 6, + 7, + 8, + 9, + 12, + 13, + 15, + t(1;1)(p32;q32), + der(1)t(1 ;8)(p 13;q 11), + del(2)(p 11), + del(2)(q22), + del(2)(q22), + del(3)(p13), + i(5p),t(8;14)(q24;q24), + der(8)t(8;14) (q24;q24), + del(10)(p12),der(11)t(11;15)(p15;q11),t(16;?)(pl 3;?),t(16;?)(q24;?),der dic(17) (17pter-*cen-*l 7q25::hsr::l 7q25-*cen-*17pter), + t(19;?)(p13;?), + der dic(20)(2Opter--*cen--~ 20q12::hsr::20q12--~cen-~20pter), + mar. The DNA index was 1.59. The finding in these leiomyosarcomas of rearrangements of the same regions of chromosomes 12 and 14 that are involved in the tumor-specific t(12;14)(q14-15;q23-24) of uterine leiomyoma indicates that the same genes in 12q and 14q might be important in the pathogenesis of benign and malignant smaoth muscle tumors.
INTRODUCTION L e i o m y o s a r c o m a r e p r e s e n t s o n e t e n t h of soft t i s s u e s a r c o m a s a n d o n e f o u r t h of u t e r i n e s a r c o m a s [1, 2]. C y t o g e n e t i c s t u d i e s of l e i o m y o s a r c o m a h a v e s h o w n c h r o m o s o m e a n o m a l i e s i n o n l y 17 t u m o r s f r o m d i f f e r e n t o r g a n s [ 3 - 1 4 ] . T h e k a r y o t y p i c p i c t u r e is d i v e r s e ; n o t u m o r - s p e c i f i c a b n o r m a l i t i e s h a v e b e e n i d e n t i f i e d . W e p r e s e n t t w o leiomyosarcomas, one subcutaneous and one uterine, both with complex clonal chromosome anomalies.
From the Departments of Clinical Genetics (M. N, N. M., S. H., F. M.), Orthopedics (A. R.), Clinical Pathology (H. W.), and Oncology (B. B.), University Hospital, Lund, and the Department of Gynecology (G. H.), Central Hospital, Helsingborg, Sweden. Address reprint requests to: Mef Nilbert, Department of Clinical Genetics, Lund University Hospital, S-221 85 Lund, Sweden. Received October 24, 1989; accepted January 8, 1999.
217 © 1990 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 18010
Cancer Genet Cytogenet 48:217-223 (1990) 0165-4608/90/$03.50
218
M. Nilbert et al.
CASE HISTORIES
Case 1
A 78-year-old, previously healthy man presented with a subcutaneous tumor, noticed for 2 years, of the right knee. Fine-needle aspiration cytology revealed a high-grade sarcoma. A solid 3-cm tumor was w i d e l y excised. Histopathology showed a malignant m e s e n c h y m a l tumor with variable cellularity, atypical mitotic figures, s p i n d l e - s h a p e d cells, and p r o m i n e n t nuclear p l e o m o r p h i s m (Fig. la). The mitotic rate was 7 mitoses per 10 h i g h - p o w e r fields. In some areas, the tumor had extensive hyalinization and focal calcifications. The tumor cells were positive for desmin and muscle-specific actin by the indirect peroxidase method [15]. The diagnosis was poorly differentiated leiomyosarcoma, grade IV on a four-grade scale. Chest radiographs at time of operation showed a 2-cm nodule in the right lung. At latest follow-up, 12 months after operation, there were no signs of local recurrence, but the chest nodule had increased slightly and thus, was probably a metastasis.
Case 2
A 75-year-old, p r e v i o u s l y healthy w o m a n presented with p o s t m e n o p a u s a l bleeding. Curretage revealed malignant cells, and a total hysterectomy with bilateral salpingoo o p h o r e c t o m y was performed. A partly necrotic, p o l y p o u s tumor with a 4-cm base infiltrated one third of the uterine wall. Histopathology revealed a cell-rich tumor with p r o m i n e n t vascularity, nuclear p l e o m o r p h i s m , and tumor giant cells (Fig. lb). In some areas, 3 0 - 4 0 mitoses, m a n y of them atypical, were observed per 10 highp o w e r fields. I m m u n o h i s t o c h e m i s t r y by the indirect peroxidase method was positive for d e s m i n and muscle-specific actin [15]. The diagnosis was poorly differentiated leiomyosarcoma, grade IV. Postoperative radio- and c h e m o t h e r a p y was given. At follow-up, 3 months after operation, there were no signs of local or distant tumor.
CYTOGENETIC METHODS
Fresh t u m o r tissue was m i n c e d and disaggregated in 0.8% collagenase for 3 - 4 hours. Short-term cultures were initiated on glass chamber slides in RPMI 1640 m e d i u m s u p p l e m e n t e d with 17% fetal calf serum, glutamine, and antibiotics [16]. The in situ preparations were harvested after 5 - 1 0 days. Wright stain and CBG staining were used for G- and C-banding. F l o w cytometric DNA analysis was performed according to Baldetorp et al. [17]. Briefly, the tumor tissue was m e c h a n i c a l l y disaggregated in a detergent nuclear isolation m e d i u m containing p r o p i d i u m i o d i d e and RNase. After staining, the DNA content of 15,000 nuclei per s a m p l e was a n a l y z e d in a cytofluorograph (Ortho, system 50H). Chicken and trout red blood cells were used as internal standards for the calculation of the DNA indices.
RESULTS
Case 1
Of the 19 cells analyzed, one was karyotypically normal, one had the karyotype 4 6 , X , - X , ÷ 7, and 17 had c o m p l e x chromosome rearrangements. Of these, 11 were h y p e r t r i p l o i d with the karyotype 70-80,XY, + X, + Y, + 1, + 1, + 2, + 2, + 3, + 3, + 4,
a
b Figure 1 Poorly differentiated leiomyosarcomas (a] (case 1) subcutaneous tumor with huclear pleomorphism (H&E x loo),and (b) (case 2) uterine tumor with high mitotic rate an tumor giant cells (H&E x 100).
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F i g u r e 2 Karyotype from case 1: 7 1 , X Y , + X . + Y , + 3 , + 4 , + 4 . + 7 , + 7 , + 8 , + 8 , + 1 0 , + 1 5 , + 1 5 , + 16, + 19, + 21, + 21. + 22,t(?;5){5;21 )(?;q35p11 ;ql 1 ),t(?;5)(5;21 )(?;q35pl I ;ql 1 ),der(13]t(12;13) (q13;q22),der(13)t(12;13)(q13;q22),der(14)t(9;14)(p11;p11 ), + 14p +, + t(2t);?)(q13;?),t(20:?) (q13;?), + 3 mar. The chromosomes within parentheses are selected from a polyploidized metaphase. M1 represents a del{11)(q22), whereas M2 is an unidentified marker. The three markers above are nonclonal. Arrowheads indicate breakpoints. + 4 , + 7, + 7, + 8 , + 8 , + 9 , + 1 0 , + 15, + 15, + 16, + 1 6 , + 18, + 1 9 , + 20, + 21, + 21, + 22, + 22,t(?;5)(5;21)(?;q35pll;q11),t(?;5)(5;21)(?;q35p11;q11), + del(11)(q22),der(13) t(12;13)(q13;q22),der(14)t(9;14)(pll;p11), + 14p + , + t(20;?)(q13;?), + t(20;?)(q13;?), + 2 mar (Fig. 2). Six cells had c h r o m o s o m e n u m b e r s b e t w e e n 120 and 150 and the s a m e markers that w e r e f o u n d in the h y p e r t r i p l o i d clone. T h e s e cells t h u s r e p r e s e n t a p o l y p l o i d i z e d v e r s i o n of the first clone. T e l o m e r i c associations i n v o l v i n g different c h r o m o s o m e s w e r e o b s e r v e d in m o s t cells. A marker ring was f o u n d in one m e t a p h a s e . D N A c y t o m e t r y gave an i n d e x of 1.76. Case 2
Of the 23 m e t a p h a s e s k a r y o t y p e d , t w o w e r e normal. The r e m a i n i n g 21, all of w h i c h had m u l t i p l e a n o m a l i e s , w e r e in the h y p o t r i p l o i d region (Fig. 3). The k a r y o t y p e was 6 1 - 6 7 , X , - X, + 1, + 3, + 5, + 6, + 7, + 8, + 9, + 12, + 13, + 15, + t(1;1)(p32;q32), + der(1) t(1;8)(p13;q11), + del(2)(p11), + del(2)(q22), + del(2)(q22), + del(3)(p13), + i(5p),t(8;14) (q24;q24), + der(8)t(8;14)(q24;q24), + del(10)(p12),der(11)t(11;15)(p15;q11),t(16;?) (p13;?),t(16;?)(q24;?),der dic(17)(17pter--~cen-~17q25 ::hsr::17q251~cen--~ 17pter), t(19;?)(p13;?), + der dic(20)(20pter-~cen-*20q12::hsr::20q12--~cen--~20pter), + mar. The structural r e a r r a n g e m e n t s of c h r o m s o m e s 1 and 16 wer~ c o n f i r m e d by C-banding. The D N A i n d e x was 1.59. DISCUSSION
F l o w c y t o m e t r i c analysis of the D N A ~ : o n t e n t y i e l d e d D N A i n d i c e s in good a g r e e m e n t w i t h the s t e m line c h r o m o s o m e n u m b e r s . H o w e v e r , in case 1 o n l y one peak, corres p o n d i n g t~ a D N A i n d e x of 1.76, was o b s e r v e d in the DNA histogram, a l t h o u g h an
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Figure 3 Karyotype from case 2: 65,X,-X,+5,+6,+7,+,9+,+12,+13,+13,+22,t(1;1) (p32;q32), + der(1)t(1;8)(pl3;qll), + del(2)(pll), + del(2)(q22), + del(2)(q22), + del(3)(pl3), + i(5p),t(8;14)(q24;q24), + der(8)t(8;14)(q24;q24), + del(10)(p12),der(11)t(11;15)(p15;q11),t(16;?) (p13;?),t(16;?)(q24;?),der dic(17)(17pter--~cen--~17q25::hsr::17q25--~cen--*17pter),+ t(19;?/ (p13;?),der dic(20)(20pter-*cen--,20q12::hsr::20q12-*cen--~20pter), + 3mar. Of the three markers in the right margin, only the one to the left was c~onal. Arrowheads indicate breakpoints.
additional clone with 120-150 chromosomes was detected cytogenetically. Either this massively rearranged population must have arisen through polyploidization in vitro, or it was present in vivo but was too small to be detected by flow cytometry. The two leiomyosarcomas presented here both showed extensive numerical and structural rearrangements with no similarities to the der(X)t(X;4)(:Xq26--*cen--~ Xp22::4q23--~4qter),del(4)(q23), and t(10;17)(q22.1;p13) previously described in leiomyosarcomas of soft tissue and uterus [9, 10]. The uterine leiomyosarcoma of case 2 had a der(11)t(11;15)(p15;q11). Rearrangements of 11p have been reported in three other leiomyosarcomas: as t(2;11;19) (q22;p13;q13.3) in a prostatic tumor [6], as del(11)(p13) in a retroperitoneal tumor [11], and as der(11)t(9;11)(:11p12-*11q25::9q12-*9qter) in a ventricular leiomyosarcoma [12]. Changes of 11p, possibly involving an inactivation mutation or loss of chromosomal material, may therefore be an important step in leiomyosarcoma pathogenesis. In addition to the der(11)t(11;15)(p15;q11), case 2 also had a 19p + marker chromosome. A cytogenetically similar 19p + marker has, often together with different anomalies of 11p, recantly been described in malignant fibrous histiocytoma [18], a tumor type with many similarities to leiomyosarcoma [1]. Telomeric associations involving different chromosomes were observed in most of the cells in case 1, and this p h e n o m e n o n too has been described in malignant fibrous histiocytoma [19]. Case 2 had an i(5p), an anomaly that has been found together with other changes in a prostatic leiomyosarcoma [5]. This anomaly has been reported as a primary change in bladder tumors and as a secondary change in tumors of the breast, lung, large bowel, uterine cervix, and ovary [20]. Chromosomal studies of uterine leiomyoma [21-25] have identified three cytogenetic subgroups characterized by t(12;14)(q14-15;q23-24),del(7)(q21.2q31.2), and
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M. Nilbert et al. + 12. Case 1 had a der(13)t(12;13)(q13;q22), and case 2 had a t(8;14)(q24;q24), i.e., rearrangements involving the same segments of chromosomes 12 and 14 that are rearranged in leiomyoma. The genes in 12q and 14q involved in the t(12;14) (q14-15;q23-24) are u n k n o w n [26]. The finding of similar breakpoints in the leiomyosarcomas reported here, albeit without cytogenetic evidence of any exchange of genetic material between 12q and 14q, indicates that the same loci may be of importance also in the development of leiomyosarcoma.
This work was supported by grants from the Swedish Cancer Society, the Swedish Work Environment Fund, the Swedish Society for Medical Research, the Lund University Medical Faculty, and the JAP Foundation for Medical Research.
REFERENCES 1. Enzinger FM, Weiss SW (1988): Soft Tissue Tumors. C. V. Mosby, St Louis, pp. 269-299, 402-421. 2. Zaloudek CJ, Norris HJ (1987): Mesenchymal tumors of the uterus. In: Blaustein's Pathology of the Female Genital Tract, 3rd Ed., RJ Kurman, ed. Springer-Verlag, New York, pp. 373-408. 3. Mark J (1976): G-band analyses of a human intestinal leiomyosarcoma. APMIS Sect A 84:538-540. 4. Becher R, Wake N, Gibas Z, Ochi H, Sandberg AA (1984): Chromosome changes in soft tissue sarcomas. JNC[ 72:823-831. 5. Atkin NB, Baker CB (1985): Chromosome study of five cancers of the prostate. Hum Genet 70:359-364. 6. Limon J, Dal Cin P, Sandberg AA (1986J: Cytogenetic findings in a primary leiomyosarcoma of the prostate. Cancer Genet Cytogenet 22:159-167. 7. Teyssier JR (1987): Nonrandom chromosomal changes in human solid tumors: Application of an improved culture method. JNCI 79:1189-1198. 8. Dal Cin P, Boghosian L, Sandberg AA (1988): Cytogenetic findings in leiomyosarcoma of the small bowel. Cancer Genet Cytogenet 30:285-288. 9. Dal Cin P, Boghosian L, Crickard K, Sandberg AA (1988): t(10;17) as the sole chromosome change in uterine leiomyosarcoma. Cancer Genet Cytogenet 32:263-266. 10. Nilbert M, Mandahl N, Helm S, Rydholm A, Will~n H, Akerman M, Mitelman F (1988): Chromosome abnormalities in leiomyosarcomas. Cancer Genet Cytogenet 34:209-218. 11. Salt S, Dal Cin P, Sandberg AA (19881: Consistent chromosome changes in leiomyosarcoma. Cancer Genet Cytogenet 35:47-50. 12. Mark J, Wedell B, Dahlenfors R, Havel G (1989): Cytogenetic observations in a human gastric leiomyosarcoma. Cancer Genet Cytogenet 37:215-220. 13. Boghosian L, Dal Cin P, Turc-Carel C, Rao U, Karakousis C, Salt SJ, Sandberg AA (1989): Three possible cytogenetic subgroups of leiomyosarcoma. Cancer Genet Cytogenet 43:39-49. 14. Nilbert M, Jin Y, Helm S, Mandahl N, Flod6rus U-M, Will6n H, Mitelman F (1990): Chromosome rearrangements in two uterine sarcomas. Cancer Genet Cytogenet 44:27-35. 15. Roholl PJM, De Jong ASH, Ramaekers FCS (1985): Application of markers in the diagnosis of soft tissue tumors. Histopathology 9:1019-1035. 16. Mandahl N, Helm S, Arheden K, Rydholm A, Will6n H, Mitelman F (1988): Three major cytogenetic subgroups can be identified among chromosomally abnormal solitary lipomas. Hum Genet 79:203-208. 17. Baldetorp B, Dalberg M, Hoist U, Lindgren G (1989): Statistical evaluation of cell kinetic data from DNA flow cytometry (FCM) by the EM-algorithm. Cytometry 10:695-705. 18. Mandahl N, Helm S, Will6n H, Rydholm A, Eneroth M, Nilbert M, Kreicbergs A, Mitelman F (1989): Characteristic karyotypic anomalies identify subtypes of malignant fibrous histiocytoma. Genes Chrom Cancer 1:9-14. 19. Mandahl N, Helm S, Arheden K, Rydholm A, Will6n H, Mitelman F (1988): Rings, dicentrics, and telomeric associations in histiocytomas. Cancer Genet Cytogenet 30:23-33.
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20. Mitelman F (1988): Catalog of Chromosome Aberrations in Cancer, 3rd Ed., Alan R. Liss, New York. 21. Heim S, Nilbert M, Vanni R, Flod~rus U-M, Mandahl N, Liedgren S, Lecca U, Mitelman F (1988): A specific translocation, t(12;14)(q14-15;q23-24}, characterizes a subgroup of uterine leiomyomas. Cancer Genet Cytogenet 32:13-17. 22. Turc-Carel C, Dal Cin P, Boghosian L, Terk-Zakarian J, Sandberg AA (1988): Consistent breakpoint in region 14q22-q24 in uterine leiomyoma. Cancer Genet Cytogenet 32:25-31. 23. Nilbert M, Heim S, Mandahl N, Flod~rus U-M, Will~n H, Mitelman F (1989): Karyotypic rearrangements in 20 uterine leiomyomas. Cytogenet Cell Genet 49:300-304. 24. 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. 25. Nilbert M, Heim S, Mandahl N, Flod~rus U-M, Will~n H, Mitelman F (1990): Trisomy 12 in uterine leiomyoma--a new cytogenetic subgroup. Cancer Genet Cytogenet, 45:63-66. 26. Arheden K, Nilbert M, Heim S, Mandahl N, Mitelman F (1989): No amplification or rearrangement of INT1, GLI, or COL2A1 in uterine leiomyomas with t(12;14)(q14-15;q23-24). Cancer Genet Cytogenet 39:195-210.
ABSTRACT: Cytogenetic investigation of short-term cultures from two leiomyosarcomas revealed complex karyotypic changes in both cases. The first tumor, a subcutaneous leiomyosarcoma of the knee, had the karyotype 7 0 - 8 0 , X Y , + X , + Y , + l , + l , + 2 , + 2 , + 3 , + 3 , + 4 , + 4 , + 7 , + 7 , + 8 , + 8 , + 9, +10, +15, +15, +16, +16, +18, +19, + 20, + 21, + 21, + 22, + 22,t(?;5)(5;21)(?;q35p11;q11), t(?;5)(5;21)(?;q35pl 1;ql 1), + del(11)(q22),der(13)t(12;13)(q13;q22),der(14)t(9;14)(pl 1;pl 1), + 1 4 p + , + t(20;?)(q13;?), +t(20;?)(q13;?), + 2 mar. A polyploidized clone with 120-150 chromosomes was also abserved. DNA flow cytometry revealed only one abnormal peak, corresponding to a DNA index of 1.76. The other tumor, a uterine leiomyosarcoma, had the karyotype 61-67, X, - X , + 1, + 3, + 5, + 6, + 7, + 8, + 9, + 12, + 13, + 15, + t(1;1)(p32;q32), + der(1)t(1 ;8)(p 13;q 11), + del(2)(p 11), + del(2)(q22), + del(2)(q22), + del(3)(p13), + i(5p),t(8;14)(q24;q24), + der(8)t(8;14) (q24;q24), + del(10)(p12),der(11)t(11;15)(p15;q11),t(16;?)(pl 3;?),t(16;?)(q24;?),der dic(17) (17pter-*cen-*l 7q25::hsr::l 7q25-*cen-*17pter), + t(19;?)(p13;?), + der dic(20)(2Opter--*cen--~ 20q12::hsr::20q12--~cen-~20pter), + mar. The DNA index was 1.59. The finding in these leiomyosarcomas of rearrangements of the same regions of chromosomes 12 and 14 that are involved in the tumor-specific t(12;14)(q14-15;q23-24) of uterine leiomyoma indicates that the same genes in 12q and 14q might be important in the pathogenesis of benign and malignant smaoth muscle tumors.
INTRODUCTION L e i o m y o s a r c o m a r e p r e s e n t s o n e t e n t h of soft t i s s u e s a r c o m a s a n d o n e f o u r t h of u t e r i n e s a r c o m a s [1, 2]. C y t o g e n e t i c s t u d i e s of l e i o m y o s a r c o m a h a v e s h o w n c h r o m o s o m e a n o m a l i e s i n o n l y 17 t u m o r s f r o m d i f f e r e n t o r g a n s [ 3 - 1 4 ] . T h e k a r y o t y p i c p i c t u r e is d i v e r s e ; n o t u m o r - s p e c i f i c a b n o r m a l i t i e s h a v e b e e n i d e n t i f i e d . W e p r e s e n t t w o leiomyosarcomas, one subcutaneous and one uterine, both with complex clonal chromosome anomalies.
From the Departments of Clinical Genetics (M. N, N. M., S. H., F. M.), Orthopedics (A. R.), Clinical Pathology (H. W.), and Oncology (B. B.), University Hospital, Lund, and the Department of Gynecology (G. H.), Central Hospital, Helsingborg, Sweden. Address reprint requests to: Mef Nilbert, Department of Clinical Genetics, Lund University Hospital, S-221 85 Lund, Sweden. Received October 24, 1989; accepted January 8, 1999.
217 © 1990 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 18010
Cancer Genet Cytogenet 48:217-223 (1990) 0165-4608/90/$03.50
218
M. Nilbert et al.
CASE HISTORIES
Case 1
A 78-year-old, previously healthy man presented with a subcutaneous tumor, noticed for 2 years, of the right knee. Fine-needle aspiration cytology revealed a high-grade sarcoma. A solid 3-cm tumor was w i d e l y excised. Histopathology showed a malignant m e s e n c h y m a l tumor with variable cellularity, atypical mitotic figures, s p i n d l e - s h a p e d cells, and p r o m i n e n t nuclear p l e o m o r p h i s m (Fig. la). The mitotic rate was 7 mitoses per 10 h i g h - p o w e r fields. In some areas, the tumor had extensive hyalinization and focal calcifications. The tumor cells were positive for desmin and muscle-specific actin by the indirect peroxidase method [15]. The diagnosis was poorly differentiated leiomyosarcoma, grade IV on a four-grade scale. Chest radiographs at time of operation showed a 2-cm nodule in the right lung. At latest follow-up, 12 months after operation, there were no signs of local recurrence, but the chest nodule had increased slightly and thus, was probably a metastasis.
Case 2
A 75-year-old, p r e v i o u s l y healthy w o m a n presented with p o s t m e n o p a u s a l bleeding. Curretage revealed malignant cells, and a total hysterectomy with bilateral salpingoo o p h o r e c t o m y was performed. A partly necrotic, p o l y p o u s tumor with a 4-cm base infiltrated one third of the uterine wall. Histopathology revealed a cell-rich tumor with p r o m i n e n t vascularity, nuclear p l e o m o r p h i s m , and tumor giant cells (Fig. lb). In some areas, 3 0 - 4 0 mitoses, m a n y of them atypical, were observed per 10 highp o w e r fields. I m m u n o h i s t o c h e m i s t r y by the indirect peroxidase method was positive for d e s m i n and muscle-specific actin [15]. The diagnosis was poorly differentiated leiomyosarcoma, grade IV. Postoperative radio- and c h e m o t h e r a p y was given. At follow-up, 3 months after operation, there were no signs of local or distant tumor.
CYTOGENETIC METHODS
Fresh t u m o r tissue was m i n c e d and disaggregated in 0.8% collagenase for 3 - 4 hours. Short-term cultures were initiated on glass chamber slides in RPMI 1640 m e d i u m s u p p l e m e n t e d with 17% fetal calf serum, glutamine, and antibiotics [16]. The in situ preparations were harvested after 5 - 1 0 days. Wright stain and CBG staining were used for G- and C-banding. F l o w cytometric DNA analysis was performed according to Baldetorp et al. [17]. Briefly, the tumor tissue was m e c h a n i c a l l y disaggregated in a detergent nuclear isolation m e d i u m containing p r o p i d i u m i o d i d e and RNase. After staining, the DNA content of 15,000 nuclei per s a m p l e was a n a l y z e d in a cytofluorograph (Ortho, system 50H). Chicken and trout red blood cells were used as internal standards for the calculation of the DNA indices.
RESULTS
Case 1
Of the 19 cells analyzed, one was karyotypically normal, one had the karyotype 4 6 , X , - X , ÷ 7, and 17 had c o m p l e x chromosome rearrangements. Of these, 11 were h y p e r t r i p l o i d with the karyotype 70-80,XY, + X, + Y, + 1, + 1, + 2, + 2, + 3, + 3, + 4,
a
b Figure 1 Poorly differentiated leiomyosarcomas (a] (case 1) subcutaneous tumor with huclear pleomorphism (H&E x loo),and (b) (case 2) uterine tumor with high mitotic rate an tumor giant cells (H&E x 100).
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F i g u r e 2 Karyotype from case 1: 7 1 , X Y , + X . + Y , + 3 , + 4 , + 4 . + 7 , + 7 , + 8 , + 8 , + 1 0 , + 1 5 , + 1 5 , + 16, + 19, + 21, + 21. + 22,t(?;5){5;21 )(?;q35p11 ;ql 1 ),t(?;5)(5;21 )(?;q35pl I ;ql 1 ),der(13]t(12;13) (q13;q22),der(13)t(12;13)(q13;q22),der(14)t(9;14)(p11;p11 ), + 14p +, + t(2t);?)(q13;?),t(20:?) (q13;?), + 3 mar. The chromosomes within parentheses are selected from a polyploidized metaphase. M1 represents a del{11)(q22), whereas M2 is an unidentified marker. The three markers above are nonclonal. Arrowheads indicate breakpoints. + 4 , + 7, + 7, + 8 , + 8 , + 9 , + 1 0 , + 15, + 15, + 16, + 1 6 , + 18, + 1 9 , + 20, + 21, + 21, + 22, + 22,t(?;5)(5;21)(?;q35pll;q11),t(?;5)(5;21)(?;q35p11;q11), + del(11)(q22),der(13) t(12;13)(q13;q22),der(14)t(9;14)(pll;p11), + 14p + , + t(20;?)(q13;?), + t(20;?)(q13;?), + 2 mar (Fig. 2). Six cells had c h r o m o s o m e n u m b e r s b e t w e e n 120 and 150 and the s a m e markers that w e r e f o u n d in the h y p e r t r i p l o i d clone. T h e s e cells t h u s r e p r e s e n t a p o l y p l o i d i z e d v e r s i o n of the first clone. T e l o m e r i c associations i n v o l v i n g different c h r o m o s o m e s w e r e o b s e r v e d in m o s t cells. A marker ring was f o u n d in one m e t a p h a s e . D N A c y t o m e t r y gave an i n d e x of 1.76. Case 2
Of the 23 m e t a p h a s e s k a r y o t y p e d , t w o w e r e normal. The r e m a i n i n g 21, all of w h i c h had m u l t i p l e a n o m a l i e s , w e r e in the h y p o t r i p l o i d region (Fig. 3). The k a r y o t y p e was 6 1 - 6 7 , X , - X, + 1, + 3, + 5, + 6, + 7, + 8, + 9, + 12, + 13, + 15, + t(1;1)(p32;q32), + der(1) t(1;8)(p13;q11), + del(2)(p11), + del(2)(q22), + del(2)(q22), + del(3)(p13), + i(5p),t(8;14) (q24;q24), + der(8)t(8;14)(q24;q24), + del(10)(p12),der(11)t(11;15)(p15;q11),t(16;?) (p13;?),t(16;?)(q24;?),der dic(17)(17pter--~cen-~17q25 ::hsr::17q251~cen--~ 17pter), t(19;?)(p13;?), + der dic(20)(20pter-~cen-*20q12::hsr::20q12--~cen--~20pter), + mar. The structural r e a r r a n g e m e n t s of c h r o m s o m e s 1 and 16 wer~ c o n f i r m e d by C-banding. The D N A i n d e x was 1.59. DISCUSSION
F l o w c y t o m e t r i c analysis of the D N A ~ : o n t e n t y i e l d e d D N A i n d i c e s in good a g r e e m e n t w i t h the s t e m line c h r o m o s o m e n u m b e r s . H o w e v e r , in case 1 o n l y one peak, corres p o n d i n g t~ a D N A i n d e x of 1.76, was o b s e r v e d in the DNA histogram, a l t h o u g h an
LeiomyosarcomaCytogenetics
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Figure 3 Karyotype from case 2: 65,X,-X,+5,+6,+7,+,9+,+12,+13,+13,+22,t(1;1) (p32;q32), + der(1)t(1;8)(pl3;qll), + del(2)(pll), + del(2)(q22), + del(2)(q22), + del(3)(pl3), + i(5p),t(8;14)(q24;q24), + der(8)t(8;14)(q24;q24), + del(10)(p12),der(11)t(11;15)(p15;q11),t(16;?) (p13;?),t(16;?)(q24;?),der dic(17)(17pter--~cen--~17q25::hsr::17q25--~cen--*17pter),+ t(19;?/ (p13;?),der dic(20)(20pter-*cen--,20q12::hsr::20q12-*cen--~20pter), + 3mar. Of the three markers in the right margin, only the one to the left was c~onal. Arrowheads indicate breakpoints.
additional clone with 120-150 chromosomes was detected cytogenetically. Either this massively rearranged population must have arisen through polyploidization in vitro, or it was present in vivo but was too small to be detected by flow cytometry. The two leiomyosarcomas presented here both showed extensive numerical and structural rearrangements with no similarities to the der(X)t(X;4)(:Xq26--*cen--~ Xp22::4q23--~4qter),del(4)(q23), and t(10;17)(q22.1;p13) previously described in leiomyosarcomas of soft tissue and uterus [9, 10]. The uterine leiomyosarcoma of case 2 had a der(11)t(11;15)(p15;q11). Rearrangements of 11p have been reported in three other leiomyosarcomas: as t(2;11;19) (q22;p13;q13.3) in a prostatic tumor [6], as del(11)(p13) in a retroperitoneal tumor [11], and as der(11)t(9;11)(:11p12-*11q25::9q12-*9qter) in a ventricular leiomyosarcoma [12]. Changes of 11p, possibly involving an inactivation mutation or loss of chromosomal material, may therefore be an important step in leiomyosarcoma pathogenesis. In addition to the der(11)t(11;15)(p15;q11), case 2 also had a 19p + marker chromosome. A cytogenetically similar 19p + marker has, often together with different anomalies of 11p, recantly been described in malignant fibrous histiocytoma [18], a tumor type with many similarities to leiomyosarcoma [1]. Telomeric associations involving different chromosomes were observed in most of the cells in case 1, and this p h e n o m e n o n too has been described in malignant fibrous histiocytoma [19]. Case 2 had an i(5p), an anomaly that has been found together with other changes in a prostatic leiomyosarcoma [5]. This anomaly has been reported as a primary change in bladder tumors and as a secondary change in tumors of the breast, lung, large bowel, uterine cervix, and ovary [20]. Chromosomal studies of uterine leiomyoma [21-25] have identified three cytogenetic subgroups characterized by t(12;14)(q14-15;q23-24),del(7)(q21.2q31.2), and
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M. Nilbert et al. + 12. Case 1 had a der(13)t(12;13)(q13;q22), and case 2 had a t(8;14)(q24;q24), i.e., rearrangements involving the same segments of chromosomes 12 and 14 that are rearranged in leiomyoma. The genes in 12q and 14q involved in the t(12;14) (q14-15;q23-24) are u n k n o w n [26]. The finding of similar breakpoints in the leiomyosarcomas reported here, albeit without cytogenetic evidence of any exchange of genetic material between 12q and 14q, indicates that the same loci may be of importance also in the development of leiomyosarcoma.
This work was supported by grants from the Swedish Cancer Society, the Swedish Work Environment Fund, the Swedish Society for Medical Research, the Lund University Medical Faculty, and the JAP Foundation for Medical Research.
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20. Mitelman F (1988): Catalog of Chromosome Aberrations in Cancer, 3rd Ed., Alan R. Liss, New York. 21. Heim S, Nilbert M, Vanni R, Flod~rus U-M, Mandahl N, Liedgren S, Lecca U, Mitelman F (1988): A specific translocation, t(12;14)(q14-15;q23-24}, characterizes a subgroup of uterine leiomyomas. Cancer Genet Cytogenet 32:13-17. 22. Turc-Carel C, Dal Cin P, Boghosian L, Terk-Zakarian J, Sandberg AA (1988): Consistent breakpoint in region 14q22-q24 in uterine leiomyoma. Cancer Genet Cytogenet 32:25-31. 23. Nilbert M, Heim S, Mandahl N, Flod~rus U-M, Will~n H, Mitelman F (1989): Karyotypic rearrangements in 20 uterine leiomyomas. Cytogenet Cell Genet 49:300-304. 24. 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. 25. Nilbert M, Heim S, Mandahl N, Flod~rus U-M, Will~n H, Mitelman F (1990): Trisomy 12 in uterine leiomyoma--a new cytogenetic subgroup. Cancer Genet Cytogenet, 45:63-66. 26. Arheden K, Nilbert M, Heim S, Mandahl N, Mitelman F (1989): No amplification or rearrangement of INT1, GLI, or COL2A1 in uterine leiomyomas with t(12;14)(q14-15;q23-24). Cancer Genet Cytogenet 39:195-210.