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Cytogenetic analysis of the mature teratoma and the choriocarcinoma component of a testicular mixed nonseminomatous germ cell tumor
Cytogenetic Analysis of the Mature Teratoma and the Choriocarcinoma Component of a Testicular Mixed Nonseminomatous Germ Cell Tumor Willem E. de Graaff, J. Wolter Oosterhuis, Bauke de Jong, Jannie van Echten-Arends, Janneke Wiersema-Buist, Heimen Schraffordt Koops, and Dirk Th. Sleijfer
We karyotyped two histologically distinct components with different metastatic behavior of a testicular nonseminomatous germ cell tumor. The two components showed an almost identical chromosomal pattern. These almost identical karyotypes of the two components with different metastatic potential suggest that the difference in biologic behavior might result from subtle differences (on microscopic or submicroscopic level) in chromosomal pattern or that these differences are predominantly epigenetically determined and depend primarily on the lineage of differentiation of the tumor component. Trophoblastic differentiation results in an aggressive, angioinvasive tumor but in developm e n t of teratoma in a tumor with low malignant potential. ABSTRACT:
INTRODUCTION
CASE REPORT
Testicular nonseminomatous germ cell tumors (TNSGCTs) usually metastasize primarily by the lymphatic route. The circulation is reached through the paralumbar lymph nodes and the thoracic duct. A well-known exception to this rule are tumors consisting of chorionic carcinoma (CH), which directly give rise to hematogenic metastases and therefore have a high metastatic potential [1]. Recently we reported that mature teratoma (MT) has a relatively low metastatic potential and therefore infrequently reaches the circulation [2]. The presence of MT in primary TNSGCTs is associated with a good prognosis [3, 4], in contrast to the presence of CH, which is reported to influence prognosis adversely [5, 6]. We separately karyotyped the histologic components MT and CH of a mixed TNSGCT. Because tumor progression and a tumor's acquisition of a more malignant behavior might result from sequential chromosomal alterations [7], comparison of the karyotypes of both components of the tumor might show whether differences in metastatic potential are associated with differences in chromosomal pattern.
A 44-year-old man presented with a tumor of the right testicle. The serum marker human chorionic gonadotropin (HCG) was elevated. After orchidectomy was performed at the referring hospital, pathologic examination showed a nonseminomatous germ cell tumor. Subsequent staging, performed at the University Hospital of Groningen, showed two metastases in the lungs. Radiographic investigations of the retroperitoneal lymph nodes were inconclusive. Therefore, a staging laparotomy was performed, during which some retroperitoneal lymph nodes were excised as a biopsy. Histologic examination showed no evidence of malignancy. After four remission-induction cycles of platinumbased chemotherapy, during which HCG level became normal, residual lesions from the lungs were removed. Pathologic examination of the surgical specimens showed necrosis and fibrosis only. Twelve months after thoracotomy, the patient is alive with no evidence of disease. Cross-section of the testis showed a tumor (measuring 5 × 5 × 4 cm), composed of three juxtaposed separate nodules. The first and biggest nodule was gray-white and contained cysts and small foci of cartilaginous tissue, microscopically MT (Fig. la). Of the two smaller nodules, the first was macroscopically hemorrhagic; the second consisted of gray-white partly viable and partly necrotic tissue. Microscopic examination of these nodules showed CH and embryonal carcinoma (EC) with small foci of yolk sac tumor respectively (Figs. lb and c). Only the components MT and CH were separately sampled for cytogenetic study. Before the tissue was cultured, frozen sections confirmed that the first lesion consisted of pure MT; the other lesion consisted
From the Department of Pathology (W. E. d. G., J. W-B), Medical Genetics (B. d. J., J. v. E-A.), Surgical Oncology (H. S. K.), and Medical Oncology (D. Th. S.), University of Groningen, and the Dr. Daniel den Hoed Cancer Center, Rotterdam, (J. W. 0.) The Netherlands. Address reprint requests to: Professor Dr. Bauke de Jong, Department of Medical Genetics, State University of Groningen, Antonius Deusinglaan 4, 9713 AW Groningen, The Netherlands. Received November 19, 1991; accepted January 29, 1992. © 1992 Elsevier SciencePublishing Co., Inc.
655 Avenueof the Americas.New York, NY 10010
67 Cancer Genet Cytogenet61:67-73 (1992) 0165-4608/92/$05.00
68
W . E . de Graaff et el. culture of about 7 days. W i t h flow cytometry, the cellular DNA content was m e a s u r e d on representative fresh-frozen samples of the MT and CH components. From paraffine m b e d d e d tissue a d d i t i o n a l s a m p l e s were taken for measurement of cellular DNA content of the EC component. Both m e t h o d s have been described p r e v i o u s l y [8]. DNA content is expressed as a DNA i n d e x (DI), i.e., the ratio b e t w e e n t u m o r cells and d i p l o i d G1 cells (a d i p l o i d cell has a DI of 1.00) [9]. Two peaks are c o n s i d e r e d significantly different if the difference in DI is more than 10% [10].
RESULTS
A
B
C Figure 1 (A) Mature teratoma component of primary tumor: completely differentiated tissue. (B) Choriocarcinoma component of primary tumor: cytotrophoblast with syncytiotrophoblastic giant cells. (C) Embryonal carcinoma component of primary tumor: completely undifferentiated tissue. of CH only. DNA flow c y t o m e t r y was performed on all three components. MATERIALS AND METHODS
M e t a p h a s e cells from the MT and CH c o m p o n e n t s were harvested for c h r o m o s o m a l analysis after separate tissue
Cellular DNA Content The DNA contents of the CH and the MT parts of the primary tumor s h o w e d a similar single a n e u p l o i d peak, respectively, w i t h DI = 1.40 (Fig. 2a) and DI = 1.39 (Fig. 2b). The paraffin-embedded tissue containing EC only showed a single small a n e u p l o i d peak, with DI of about 1.5 (Fig. 2c), not significantly different from the peaks measured in the MT and the CH component.
Karyotypes F r o m both the CH and MT part of the tumor, were analyzed 10 metaphases fully. The two c o m p o n e n t s showed an almost identical pattern. In the CH as well as in the MT component, two closely related karyotypes (karyotype A and karyotype B) with almost identical n u m e r i c a l abnormalities were observed. The karyotypes differed by only two structural abnormalities. To determine the proportion of karyotype A and karyotype B in each component, we a n a l y z e d another 50 metaphases. Table I shows the number of normal copies of different c h r o m o s o m e s of the fully a n a l y z e d metaphases and their m o d a l number. Figure 3a shows a representative karyotype A in the CH part of the tumor with the description: 6 3 , X Y , + X , + 1 , + 2, + 3, + 6, + 8, + 8,del(9)(q12), + 12, + 14, + 15, + der(16)t(9;16)(q12;q22), + 17, + 19, + 20, + 21, + 22, + mar; + 5 was also clonal. Figure 3b shows a representative karyotype B in the MT part of the t u m o r with the description: 63,XY, + X, + 1, + der(1)t(1;?)(p22;?), + 2, + 3, + 6, + 8, + 8, - 9, + 12, + 14, + 15, + der(16)t(9;16) + 17, + 19, + 20, + 21, + 22,(q12;q22), + m a r . In both karyotypes the marker appeared to involve c h r o m o s o m e 9. Karyotype A differed from karyotype B by having a del(9) instead of a der(1). The ratio A : B was 23 : 27 in the CH c o m p o n e n t and 13 : 37 in the MT c o m p o n e n t
DISCUSSION
Sequential c h r o m o s o m a l alterations, e.g., gain or loss of specific chromosomes, formation of structural chromosomal abnormalities, gene mutations, loss of heterozygosity, and point mutations in protooncogenes are a major force for more aggressive characteristics and more malignant behavior of m a n y tumors [7, 11-13]. The CH and MT c o m p o n e n t s of TNSGCTs differ in metastatic potential. TNSGCTs u s u a l l y metastasize p r i m a r i l y by the l y m p h a t i c route. The circulation is reached through the p a r a l u m b a r
Cytogenetics of a Mixed N o n s e m i n o m a
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Figure 2 (A) DNA flow graph of choriocarcinoma component. (B) DNA flow graph of mature teratoma component. (C) DNA flow graph of embryonal carcinoma component.
l y m p h nodes and thoracic duct [14], but CH directly gives rise to hematogenic metastases and therefore has high metastatic potential [1]. MT has relatively low metastatic potential and infrequently reaches the circulation [2]. Evidence for a difference in metastatic potential of CH and MT is also d e m o n s t r a t e d by the case we describe. No evidence of retroperitoneal l y m p h n o d e metastases was noted. After c h e m o t h e r a p y the lung metastases s h o w e d o n l y necrosis and fibrosis, i n d i c a t i n g that before c h e m o t h e r a p y components other than teratoma had been present [2]. We c o m p a r e d the DI and karyotypes of the CH and MT
c o m p o n e n t s of a m i x e d p r i m a r y TNSGCT to determine w h e t h e r differences in biologic behavior are associated with a different c h r o m o s o m a l pattern. Both the DNA content and the c h r o m o s o m a l pattern of the two c o m p o n e n t s were almost identical. The DI of the EC c o m p o n e n t was not significantly different from that of the CH and MT components. The data suggest that the difference in biologic behavior is p r e d o m i n a n t l y epigenetically d e t e r m i n e d or m a y result from subtle differences (on m i c r o s c o p i c or submicroscopic level) in c h r o m o s o m a l pattern or a combination of genetically [15, 16] and epigenetically d e t e r m i n e d events.
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D
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21
13
6
Figure 3
15
8
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16
9
17
10
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(A) Representative karyotype of CH component. (B) Representative karyotype of MT component.
22
14
7
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4
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72
Table 1
W.E.
Copies and modal numbers
per chromosome
d e G r a a f f et al.
o f 10 f u l l y a n a l y z e d m e t a p h a s e s Chromosome
Component Choriocarcinoma
Modal Matureteratoma
Modal
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
X
Y
i(12p)
Total number
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
3 3 3 3 3 3 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2
2 2 3 3 2 2 3 2 1 2 2 2 2 2 2 2 2 3 3 2 2 2
3 3 3 3 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
2 2 2 2 2 2 2 2 2 I 2 2 2 2 2 2 2 2 2 2 2 2
4 4 4 4 4 4 4 2 3 4 4 4 4 4 4 3 4 4 4 4 4 4
I 1 I 1 1 1 I I 1 1 1 2 1 1 1 1 1 1 1 1 1 1
2 2 1 2 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
3 3 3 3 3 3 4 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2
3 3 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 3 3 3
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
3 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2
3 4 3 3 3 3 3 3 3 3 3 3 3 3 4 3 3 3 4 3 2 3
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 3 3 3 3 3 3
3 3 3 3 3 3 3 3 3 2 3 2 3 3 3 2 3 2 3 3 2 3
2 2 3 3 3 3 3 3 2 2 3 3 3 3 2 3 3 3 2 3 3 3
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
0 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 0 0 0 0 0 0 0 0 0 0 0 0 0 O 0 0 0 0 0 0 0
58 59 59 61 57 60 60 58 57 56 60 59 60 60 59 57 60 60 60 60 58 60
I n T N S G C T s , t h e a g g r e s s i v e n e s s o f t h e t u m o r p r o b a b l y is not primarily determined by karyotype evolution leading to a m o r e a g g r e s s i v e p h e n o t y p e , b u t r a t h e r b y t h e i n h e r e n t aggressiveness of the histologic component that develops a n d f i n a l l y d o m i n a t e s t h e t u m o r ; e.g., t h e a n g i o i n v a s i v e n e s s a n d p r o p e n s i t y f o r h e m a t o g e n o u s m e t a s t a s i s o f C H is i n h e r e n t w i t h t r o p h o b l a s t i c d i f f e r e n t i a t i o n [17]. The EC stem cell of different components of TNSGCTs can differentiate through an embryonic (teratoma) or extraembryonic (yolk sac tumor, CH) lineage of differentiation. This direction of differentiation may be genetically or epigenetically determined. The results of this study indicate t h a t t h e d i r e c t i o n o f d i f f e r e n t i a t i o n a p p a r e n t l y is n o t a s s o c i ated with gross differences in chromosomal pattern.
3. Sogani PC, W h i t m o r e WFJr, Herr HW, Bosl GJ, Golbey RB, W a t s o n RC, DeCosse JJ (1984): O r c h i e c t o m y a l o n e in t h e treatm e n t of clinical stage I n o n s e m i n o m a t o u s g e r m cell t u m o r of t h e testis. J Clin O n c o l 2 : 2 6 7 - 2 7 0 . 4. K l e p p O, O l s s o n AM, H e n r i k s o n H, A a s s N, Dahl O, S t e n w i g AE, P e r s s o n B-E, Cavallin-Stfihl E, Fossil SD, W a h l q v i s t L (1990): P r o g n o s t i c factors in clinical stage I n o n s e m i n o m a t o u s g e r m cell t u m o r s of t h e testis: M u l t i v a r i a t e a n a l y s i s of a pros p e c t i v e m u l t i c e n t e r study. J Clin O n c o l 8 : 5 0 9 - 5 1 8 . 5. V u g r i n D, Cvitkovic E, P o s n e r J, H a j d u S, Golbey RB (1979): N e u r o l o g i c a l c o m p l i c a t i o n s of m a l i g n a n t g e r m cell t u m o r s of testis. Biology of b r a i n m e t a s t a s e s (I). C a n c e r 4 4 : 2 3 4 9 - 2 3 5 3 . 6. J e l s m a RK, Carroll M (1989): Brain m e t a s t a s i s from n o n s e m i n o m a t o u s g e r m cell t u m o r s of t h e testis: Case report a n d review of t h e role of surgery. N e u r o s u r g e r y 2 5 : 8 1 4 - 8 1 9 . 7. Nowell PC (1990): Cytogenetics of t u m o r progression. Cancer 65:2172-2177.
T h i s s t u d y w a s s u p p o r t e d by T h e N e t h e r l a n d s C a n c e r F o u n d a t i o n , by Grant No. 88-10, a n d a g r a n t f r o m t h e Jan Kornelis de CockStichting, G r o n i n g e n , T h e N e t h e r l a n d s . T h e a u t h o r s t h a n k Dr. A. J. H. S u u r m e i j e r , D i a k o n e s s e n Z i e k e n h u i s G r o n i n g e n , t h e Netherlands, for s h a r i n g h i s material.
REFERENCES 1. O o s t e r h u i s JW (1983): T h e m e t a s t a s i s of h u m a n teratomas. In: T h e H u m a n T e r a t o m a s , I D a m j a n o v , B K n o w l e s , D SoRer, eds., H u m a n a Press, Clifton, NJ, pp. 1 3 7 - 1 7 1 . 2. De Graaff WE, O o s t e r h u i s JW, V a n der L i n d e n S, H o m a n v a n der H e i d e JN, Schraffordt K o o p s H, Sleijfer D T h (1991): Residu a l m a t u r e t e r a t o m a after c h e m o t h e r a p y for n o n s e m i n o m a t o u s g e r m cell t u m o r s of t h e testis occurs significantly less often in l u n g t h a n in retroperitoneal l y m p h n o d e m e t a s t a s e s . J U r o g e n Pathol 1:75-81.
8. O o s t e r h u i s JW, De Jong B, C o r n e l i s s e CJ, M o l e n a a r WM, Meiri n g A, I d e n b u r g VJS, Schraffordt K e e p s H, Sleijfer D T h (1986): K a r y o t y p i n g a n d DNA-flow c y t o m e t r y of m a t u r e r e s i d u a l terat o m a after i n t e n s i v e c h e m o t h e r a p y of d i s s e m i n a t e d n o n - s e m i n o m a t o u s g e r m cell t u m o r of t h e testis: a report of two cases. C a n c e r Genet C y t o g e n e t 2 2 : 1 4 9 - 1 5 7 . 9. H i d d e m a n n W, S c h u m a n n J, A n d r e e f f M, Barlogie B, H e r m a n CJ, Leif RC, Mayall BH, M u r p h y RF, S a n d b e r g A A (1984): C o n v e n t i o n o n n o m e n c l a t u r e for D N A cytometry. C y t o m e t r y 5:445-446. 10. O o s t e r h u i s JW, C a s t e d o SMMJ, De Jong B, Cornelisse CJ, D a m A, Sleijfer DTh, Schraffordt K e e p s H (1989): Ploidy of p r i m a r y g e r m cell t u m o r s of t h e testis. P a t h o g e n e t i c a n d clinical relevance. Lab Invest 6 0 : 1 4 - 2 0 . 11. H e p p n e r GH (1984): 44:2259-2265.
Tumor
heterogeneity.
Cancer
Res
12. N o w e l l PC (1986): M e c h a n i s m s of t u m o r progression. Cancer Res 4 6 : 2 2 0 3 - 2 2 0 7 . 13. Killion JJ, Fidler IJ (1989): T h e biology of t u m o r metastasis. Semin Oncol 16:106-115.
Cytogenetics of a Mixed N o n s e m i n o m a
14. Peckham MJ (1981): Investigation and staging: general aspects and staging classification. In: The Management of Testicular Tumours, MJ Peckham, ed., Edward Arnold, London, pp. 89-101. 15. Delozier-Blanchet CD, Walt H, Engel E, Vuagnat P (1987): Cytogenetic studies of human testicular germ cell tumours. Int J Androl 10:69-77.
73
16. Bosl GJ, Dmitrovsky E, Reuter VE, Samaniego F, Rodriguez E, Geller NL, Chaganti RSK (1989): Isochromosome of chromosome 12: Clinically useful marker for male germ cell tumors. J Natl Cancer Inst 81:1874-1878. 17. Ramsey EM, Houston ML, Harris JWS (1976): Interaction of the trophoblast and maternal tissues in three closely related primate species. Am J Obstet Gynecol 124:647.
We karyotyped two histologically distinct components with different metastatic behavior of a testicular nonseminomatous germ cell tumor. The two components showed an almost identical chromosomal pattern. These almost identical karyotypes of the two components with different metastatic potential suggest that the difference in biologic behavior might result from subtle differences (on microscopic or submicroscopic level) in chromosomal pattern or that these differences are predominantly epigenetically determined and depend primarily on the lineage of differentiation of the tumor component. Trophoblastic differentiation results in an aggressive, angioinvasive tumor but in developm e n t of teratoma in a tumor with low malignant potential. ABSTRACT:
INTRODUCTION
CASE REPORT
Testicular nonseminomatous germ cell tumors (TNSGCTs) usually metastasize primarily by the lymphatic route. The circulation is reached through the paralumbar lymph nodes and the thoracic duct. A well-known exception to this rule are tumors consisting of chorionic carcinoma (CH), which directly give rise to hematogenic metastases and therefore have a high metastatic potential [1]. Recently we reported that mature teratoma (MT) has a relatively low metastatic potential and therefore infrequently reaches the circulation [2]. The presence of MT in primary TNSGCTs is associated with a good prognosis [3, 4], in contrast to the presence of CH, which is reported to influence prognosis adversely [5, 6]. We separately karyotyped the histologic components MT and CH of a mixed TNSGCT. Because tumor progression and a tumor's acquisition of a more malignant behavior might result from sequential chromosomal alterations [7], comparison of the karyotypes of both components of the tumor might show whether differences in metastatic potential are associated with differences in chromosomal pattern.
A 44-year-old man presented with a tumor of the right testicle. The serum marker human chorionic gonadotropin (HCG) was elevated. After orchidectomy was performed at the referring hospital, pathologic examination showed a nonseminomatous germ cell tumor. Subsequent staging, performed at the University Hospital of Groningen, showed two metastases in the lungs. Radiographic investigations of the retroperitoneal lymph nodes were inconclusive. Therefore, a staging laparotomy was performed, during which some retroperitoneal lymph nodes were excised as a biopsy. Histologic examination showed no evidence of malignancy. After four remission-induction cycles of platinumbased chemotherapy, during which HCG level became normal, residual lesions from the lungs were removed. Pathologic examination of the surgical specimens showed necrosis and fibrosis only. Twelve months after thoracotomy, the patient is alive with no evidence of disease. Cross-section of the testis showed a tumor (measuring 5 × 5 × 4 cm), composed of three juxtaposed separate nodules. The first and biggest nodule was gray-white and contained cysts and small foci of cartilaginous tissue, microscopically MT (Fig. la). Of the two smaller nodules, the first was macroscopically hemorrhagic; the second consisted of gray-white partly viable and partly necrotic tissue. Microscopic examination of these nodules showed CH and embryonal carcinoma (EC) with small foci of yolk sac tumor respectively (Figs. lb and c). Only the components MT and CH were separately sampled for cytogenetic study. Before the tissue was cultured, frozen sections confirmed that the first lesion consisted of pure MT; the other lesion consisted
From the Department of Pathology (W. E. d. G., J. W-B), Medical Genetics (B. d. J., J. v. E-A.), Surgical Oncology (H. S. K.), and Medical Oncology (D. Th. S.), University of Groningen, and the Dr. Daniel den Hoed Cancer Center, Rotterdam, (J. W. 0.) The Netherlands. Address reprint requests to: Professor Dr. Bauke de Jong, Department of Medical Genetics, State University of Groningen, Antonius Deusinglaan 4, 9713 AW Groningen, The Netherlands. Received November 19, 1991; accepted January 29, 1992. © 1992 Elsevier SciencePublishing Co., Inc.
655 Avenueof the Americas.New York, NY 10010
67 Cancer Genet Cytogenet61:67-73 (1992) 0165-4608/92/$05.00
68
W . E . de Graaff et el. culture of about 7 days. W i t h flow cytometry, the cellular DNA content was m e a s u r e d on representative fresh-frozen samples of the MT and CH components. From paraffine m b e d d e d tissue a d d i t i o n a l s a m p l e s were taken for measurement of cellular DNA content of the EC component. Both m e t h o d s have been described p r e v i o u s l y [8]. DNA content is expressed as a DNA i n d e x (DI), i.e., the ratio b e t w e e n t u m o r cells and d i p l o i d G1 cells (a d i p l o i d cell has a DI of 1.00) [9]. Two peaks are c o n s i d e r e d significantly different if the difference in DI is more than 10% [10].
RESULTS
A
B
C Figure 1 (A) Mature teratoma component of primary tumor: completely differentiated tissue. (B) Choriocarcinoma component of primary tumor: cytotrophoblast with syncytiotrophoblastic giant cells. (C) Embryonal carcinoma component of primary tumor: completely undifferentiated tissue. of CH only. DNA flow c y t o m e t r y was performed on all three components. MATERIALS AND METHODS
M e t a p h a s e cells from the MT and CH c o m p o n e n t s were harvested for c h r o m o s o m a l analysis after separate tissue
Cellular DNA Content The DNA contents of the CH and the MT parts of the primary tumor s h o w e d a similar single a n e u p l o i d peak, respectively, w i t h DI = 1.40 (Fig. 2a) and DI = 1.39 (Fig. 2b). The paraffin-embedded tissue containing EC only showed a single small a n e u p l o i d peak, with DI of about 1.5 (Fig. 2c), not significantly different from the peaks measured in the MT and the CH component.
Karyotypes F r o m both the CH and MT part of the tumor, were analyzed 10 metaphases fully. The two c o m p o n e n t s showed an almost identical pattern. In the CH as well as in the MT component, two closely related karyotypes (karyotype A and karyotype B) with almost identical n u m e r i c a l abnormalities were observed. The karyotypes differed by only two structural abnormalities. To determine the proportion of karyotype A and karyotype B in each component, we a n a l y z e d another 50 metaphases. Table I shows the number of normal copies of different c h r o m o s o m e s of the fully a n a l y z e d metaphases and their m o d a l number. Figure 3a shows a representative karyotype A in the CH part of the tumor with the description: 6 3 , X Y , + X , + 1 , + 2, + 3, + 6, + 8, + 8,del(9)(q12), + 12, + 14, + 15, + der(16)t(9;16)(q12;q22), + 17, + 19, + 20, + 21, + 22, + mar; + 5 was also clonal. Figure 3b shows a representative karyotype B in the MT part of the t u m o r with the description: 63,XY, + X, + 1, + der(1)t(1;?)(p22;?), + 2, + 3, + 6, + 8, + 8, - 9, + 12, + 14, + 15, + der(16)t(9;16) + 17, + 19, + 20, + 21, + 22,(q12;q22), + m a r . In both karyotypes the marker appeared to involve c h r o m o s o m e 9. Karyotype A differed from karyotype B by having a del(9) instead of a der(1). The ratio A : B was 23 : 27 in the CH c o m p o n e n t and 13 : 37 in the MT c o m p o n e n t
DISCUSSION
Sequential c h r o m o s o m a l alterations, e.g., gain or loss of specific chromosomes, formation of structural chromosomal abnormalities, gene mutations, loss of heterozygosity, and point mutations in protooncogenes are a major force for more aggressive characteristics and more malignant behavior of m a n y tumors [7, 11-13]. The CH and MT c o m p o n e n t s of TNSGCTs differ in metastatic potential. TNSGCTs u s u a l l y metastasize p r i m a r i l y by the l y m p h a t i c route. The circulation is reached through the p a r a l u m b a r
Cytogenetics of a Mixed N o n s e m i n o m a
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G
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' '
2o]fi
Figure 2 (A) DNA flow graph of choriocarcinoma component. (B) DNA flow graph of mature teratoma component. (C) DNA flow graph of embryonal carcinoma component.
l y m p h nodes and thoracic duct [14], but CH directly gives rise to hematogenic metastases and therefore has high metastatic potential [1]. MT has relatively low metastatic potential and infrequently reaches the circulation [2]. Evidence for a difference in metastatic potential of CH and MT is also d e m o n s t r a t e d by the case we describe. No evidence of retroperitoneal l y m p h n o d e metastases was noted. After c h e m o t h e r a p y the lung metastases s h o w e d o n l y necrosis and fibrosis, i n d i c a t i n g that before c h e m o t h e r a p y components other than teratoma had been present [2]. We c o m p a r e d the DI and karyotypes of the CH and MT
c o m p o n e n t s of a m i x e d p r i m a r y TNSGCT to determine w h e t h e r differences in biologic behavior are associated with a different c h r o m o s o m a l pattern. Both the DNA content and the c h r o m o s o m a l pattern of the two c o m p o n e n t s were almost identical. The DI of the EC c o m p o n e n t was not significantly different from that of the CH and MT components. The data suggest that the difference in biologic behavior is p r e d o m i n a n t l y epigenetically d e t e r m i n e d or m a y result from subtle differences (on m i c r o s c o p i c or submicroscopic level) in c h r o m o s o m a l pattern or a combination of genetically [15, 16] and epigenetically d e t e r m i n e d events.
K
l
D
t
h
I t
&.
IM
JL
B
y
21
13
6
Figure 3
15
8
mar
16
9
17
10
18
)!!!!
11
(A) Representative karyotype of CH component. (B) Representative karyotype of MT component.
22
14
7
II
4
1=9
i ii ¸¸¸
20
t2
72
Table 1
W.E.
Copies and modal numbers
per chromosome
d e G r a a f f et al.
o f 10 f u l l y a n a l y z e d m e t a p h a s e s Chromosome
Component Choriocarcinoma
Modal Matureteratoma
Modal
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
X
Y
i(12p)
Total number
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
3 3 3 3 3 3 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2
2 2 3 3 2 2 3 2 1 2 2 2 2 2 2 2 2 3 3 2 2 2
3 3 3 3 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
2 2 2 2 2 2 2 2 2 I 2 2 2 2 2 2 2 2 2 2 2 2
4 4 4 4 4 4 4 2 3 4 4 4 4 4 4 3 4 4 4 4 4 4
I 1 I 1 1 1 I I 1 1 1 2 1 1 1 1 1 1 1 1 1 1
2 2 1 2 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
3 3 3 3 3 3 4 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2
3 3 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 3 3 3
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
3 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2
3 4 3 3 3 3 3 3 3 3 3 3 3 3 4 3 3 3 4 3 2 3
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 3 3 3 3 3 3
3 3 3 3 3 3 3 3 3 2 3 2 3 3 3 2 3 2 3 3 2 3
2 2 3 3 3 3 3 3 2 2 3 3 3 3 2 3 3 3 2 3 3 3
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
0 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 0 0 0 0 0 0 0 0 0 0 0 0 0 O 0 0 0 0 0 0 0
58 59 59 61 57 60 60 58 57 56 60 59 60 60 59 57 60 60 60 60 58 60
I n T N S G C T s , t h e a g g r e s s i v e n e s s o f t h e t u m o r p r o b a b l y is not primarily determined by karyotype evolution leading to a m o r e a g g r e s s i v e p h e n o t y p e , b u t r a t h e r b y t h e i n h e r e n t aggressiveness of the histologic component that develops a n d f i n a l l y d o m i n a t e s t h e t u m o r ; e.g., t h e a n g i o i n v a s i v e n e s s a n d p r o p e n s i t y f o r h e m a t o g e n o u s m e t a s t a s i s o f C H is i n h e r e n t w i t h t r o p h o b l a s t i c d i f f e r e n t i a t i o n [17]. The EC stem cell of different components of TNSGCTs can differentiate through an embryonic (teratoma) or extraembryonic (yolk sac tumor, CH) lineage of differentiation. This direction of differentiation may be genetically or epigenetically determined. The results of this study indicate t h a t t h e d i r e c t i o n o f d i f f e r e n t i a t i o n a p p a r e n t l y is n o t a s s o c i ated with gross differences in chromosomal pattern.
3. Sogani PC, W h i t m o r e WFJr, Herr HW, Bosl GJ, Golbey RB, W a t s o n RC, DeCosse JJ (1984): O r c h i e c t o m y a l o n e in t h e treatm e n t of clinical stage I n o n s e m i n o m a t o u s g e r m cell t u m o r of t h e testis. J Clin O n c o l 2 : 2 6 7 - 2 7 0 . 4. K l e p p O, O l s s o n AM, H e n r i k s o n H, A a s s N, Dahl O, S t e n w i g AE, P e r s s o n B-E, Cavallin-Stfihl E, Fossil SD, W a h l q v i s t L (1990): P r o g n o s t i c factors in clinical stage I n o n s e m i n o m a t o u s g e r m cell t u m o r s of t h e testis: M u l t i v a r i a t e a n a l y s i s of a pros p e c t i v e m u l t i c e n t e r study. J Clin O n c o l 8 : 5 0 9 - 5 1 8 . 5. V u g r i n D, Cvitkovic E, P o s n e r J, H a j d u S, Golbey RB (1979): N e u r o l o g i c a l c o m p l i c a t i o n s of m a l i g n a n t g e r m cell t u m o r s of testis. Biology of b r a i n m e t a s t a s e s (I). C a n c e r 4 4 : 2 3 4 9 - 2 3 5 3 . 6. J e l s m a RK, Carroll M (1989): Brain m e t a s t a s i s from n o n s e m i n o m a t o u s g e r m cell t u m o r s of t h e testis: Case report a n d review of t h e role of surgery. N e u r o s u r g e r y 2 5 : 8 1 4 - 8 1 9 . 7. Nowell PC (1990): Cytogenetics of t u m o r progression. Cancer 65:2172-2177.
T h i s s t u d y w a s s u p p o r t e d by T h e N e t h e r l a n d s C a n c e r F o u n d a t i o n , by Grant No. 88-10, a n d a g r a n t f r o m t h e Jan Kornelis de CockStichting, G r o n i n g e n , T h e N e t h e r l a n d s . T h e a u t h o r s t h a n k Dr. A. J. H. S u u r m e i j e r , D i a k o n e s s e n Z i e k e n h u i s G r o n i n g e n , t h e Netherlands, for s h a r i n g h i s material.
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Tumor
heterogeneity.
Cancer
Res
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Cytogenetics of a Mixed N o n s e m i n o m a
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73
16. Bosl GJ, Dmitrovsky E, Reuter VE, Samaniego F, Rodriguez E, Geller NL, Chaganti RSK (1989): Isochromosome of chromosome 12: Clinically useful marker for male germ cell tumors. J Natl Cancer Inst 81:1874-1878. 17. Ramsey EM, Houston ML, Harris JWS (1976): Interaction of the trophoblast and maternal tissues in three closely related primate species. Am J Obstet Gynecol 124:647.