Pathogenesis of Adult Tesficular Germ Cell Tumors A Cytogenetic Model
Bauke de Jong, J. Wolter Oosterhuis, S~rgio M. M. J. Castedo, AnneMarie Vos, and Gerard J. te Meerman
ABSTRACT: In essence, two models exist of the pathogenetic relationship between seminomas and nanseminomatous germ cell tumors (NSGCTs). In the first model, the histogenesis of s e m i n o m a s is assumed to diverge f rom that of the other testicular germ cell t umors (TGCTs) at an early stage. The neoplastic pathway of seminomas and NSGCTs is different, with limited or no crossover. The second model sugges ts that seminomas and NSGCTs have a common origin with a s ingle neoplastic pathway on which s e m i n o m a s are an intermediate stage in development of NSGCTs. Our data on the cytogenetics a n d ploidy of seminomas, combined tumors , and NSGCTs lend suppo r t to the mode l of pathogenesis of seminomas a n d NSGCTs in which all TGCTs (with the possible exception of spermatocytic seminoma and infantile yolk sac tumor) have a single origin and neoplastic pathway, with seminomas representing an intermediate stage in develop- ment of NSGCT components, as opposed to the model in which seminomas and NSGCTs develop separately. The progression of TGCTs probably proceeds f rom high to lower n u m b e r s of chromosomes and is therefore accompanied by a net loss of chromosomal material. This decrease will be the end resul t of loss of specific chromosomes, gain of s o m e other chromosomes (or part of chromosomes), a n d development of structural abnormalities.
INTRODUCTION
A model of the pathogenesis of testicular germ cell tumors (TGCTs) of adults is presented. The model is based on our own observations and those of other investiga- tors of DNA index (DI), chromosome numbers, and specific structural chromosomal abnormalities in TGCTs. Most of our cytogenetic data have been published. For a full description and discussion of these data, in particular of structural chromosomai abnormalities, the reader should consult our previous publications.
From the Departments of Human Genetics (B. d. J., S. M. M. J. C., A. M. V., G. J. t. M.), and Pathology (J. W. O., S. M. M. I. C.), University of Groningen, Groningen. The Netherlands.
Address repr in t requests to: Bauke de Jong, Ph.D., Department of Human Genetics, Ant. Deusinglaan 4, 9713 AW Groningen, The Netherlands.
S. M. M. J. C. is a m e m b e r of the staff of the Department of Medical Genetics, University of Oporto, Portugal.
Received November 17, 1989; accepted November 30, 1989.
Human germ cell tumors (GCTs) are a heterogeneous group of neoplasms. They occur in the testis, the ovary, and in extragonadal sites [1-3[. Basically, two main entities are distinguished. First are tumors composed of neoplastic germ cells, called seminoma in the testis, dysgerminoma in the ovary, and sometimes germinoma in extragonadal sites. These tumors are indistinguishable with respect to histology, immunohisto- chemistry, and ultrastructure; e.g., they all show a diffuse, membranous staining of the tumor cells with placental-like alkaline phosphates (PLAP) [unpublished observa- tions, and ref. 4]. This and many other characteristics are shared by carcinoma in situ (CIS) cells of the testis, which are the neoplastic counterparts of gonocytes [4]. For this reason Skakkebaek has proposed that tumors composed of neoplastic gonocytes be called gonocytomas [5]. The second entity is nonseminomatous GCTs (NSGCTs), which are composed of neoplastic embryonic tissues (embryonal carcinoma, imma- ture teratoma, and mature teratoma) or extraembryonic tissues (yolk sac tumor, and choriocarcinoma) or both. Nonseminomatous GCTs may have a seminoma compo- nent. According to the British classification, such tumors are called combined tumors [6]. Pathogenesis, histological composition, cytogenetics, ploidy, and degree of malig- nancy of GCTs differ depending on the anatomic site of the tumor and the age and sex of the patient.
Testicular germ cell tumors themselves are heterogeneous [see 7 and 8 for reviews of recent progress in pathology and immunohistochemistry]. Epidemiologically three groups of TGCTs can be distinguished: infantile tumors, tumors of adolescents and young adults, and tumors of elderly men. Most infantile TGCTs have the histology of yolk sac tumor [9]. Testicular germ cell tumors of adults can be divided into semino- mas (about 50%) and NSGCTs (about 40%); a smaller percentage combines seminoma and nonseminoma components. Testicular germ cell tumors of elderly men have the histology of spermatocytic seminoma. These tumors are negative for PLAP [4, Discussion] and are not composed of gonocytes, but of more mature germ cell precur- sors. They are usually diploid [see ref. 4 for review].
Most TGCTs in adults have CIS, composed of neoplastic gonocytes, as precursor lesion. Often CIS can be found in the testicular parenchyma surrounding the invasive cancer. Infantile testicular tumors lack CIS [10]. The same is true of spermatocytic seminoma, although intratubular spermatocytic seminoma has been demonstrated [11].
Thus, with the exception of infantile tumors, and spermatocytic seminoma, GCTs of the testis are supposedly derived from dysplastic germ cell precursors (gonocytes). Carcinoma in situ is the common precursor for seminomas and NSGCTs [5].
Pathogenetic Models
In essence two models show the pathogenetic relationship between seminomas and NSGCTs [see refs. 12 and 13 for recent review]. One of the models assumes that seminomas and NSGCTs derive independently from transformed (dysplastic) intratu- bular germ cells through CIS. The histogenesis of seminomas diverges from that of the other GCTs at an early stage [14-16]. The two neoplastic pathways are distinct with no (or only limited) crossover and represent either a caricature of embryogenesis or abortive spermatogenesis [12]. The other model [16, 17] suggests that all TGCTs of adults (with the possible exception of spermatocytic seminoma [11] and infantile testicular GCTs) have a single origin with one neoplastic pathway for all GCTs, on which seminomas represent an intermediate stage in development of NSGCT components. According to this view seminomas and NSGCTs might be expected to show a close relationship. We approached the problem of the pathogenetic relation- ship of seminomas and NSGCTs using DNA flow cytometry and cytogenetics as tools.
Pathogenesis of Adult Testicular Germ Cell Tumors 145
Figure I The DNA-index (D1) is shown for all testicular germ cell tumors, per tumor type. For infantile yolk sac tumors (IYST), nonseminoma (NS), and the nonseminomatous component of combined tumors (CT) the DI of the main stemlines is indicated by a closed circle; the DI of additional stemlines is indicated by an open circle. The DI of the main stemlines of seminoma (SE) and the seminomatous component of combined tumors is indicated by a closed square. The data are plotted in separate lanes for tumors of clinical stage I-IV. The median DI of the main stemlines (arrows), is for IYST, SE, combined tumors, and NS, respectively: 1.91, 1.66, 1.48, and 1.43. The median DI of the main stemlines for the seminoma and nonseminomatous components of the combined tumors (arrowheads) is 1.61 and 1.40, respectively [13].
DNA Index of Testicular Germ Cell Tumors and Carcinoma In Situ
The ploidy of TGCTs was studied by DNA flow cytometry [13]. The results are shown in Fig. 1. The median DI (diploid cells have a DI of 1) of infantile yolk sac tumor (IYST, n = 10), seminomas (n = 20), and NSGCTs (n = 36) was 1.91, 1.66, and 1.43, respectively. The observed differences are statistically significant [13]. The seminomatous and nonseminomatous components of combined tumors (n = 16) had a significantly different median DI of 1.61 and 1.40, respectively. The median DI of the seminoma components of combined tumors is not different from the median DI
146 B. de Jong et al.
Table 1 Compar ison of p lo idy of CIS and adjacent invasive GCT
aPloidies were considered different when the DI differed 10% or more. Abbreviations: DI, DNA index; C1S, carcinoma in situ; GCT. germ cell tumor; NSGCT, nonseminomatous GCT.
of pure seminomas; similarly, the median DI of the nonseminomatous components of combined tumors is not different from that of NSGCTs. Three of the 10 IYSTs and one of the 72 TGCTs of adul ts were diploid. Mult iple aneuplo id stemlines were found in four of the ten IYSTs, five of the 36 NSGCTs, and four of the nonseminomatous components of the combined tumors. Neither the seminomas nor the seminoma
components of combined tumors had mul t ip le aneuplo id stemlines. Most of the combined tumors had mul t ip le aneuplo id stemlines.
Ten of 13 combined tumors, in which both components could be separately mea- sured, showed different s temlines between the seminomatous and nonseminomatous components . Four of six spermatocyt ic seminomas were d ip lo id and two were perite- t raploid.
In 12 seminomas and 23 NSGCTs, the p lo idy of CIS of the adul t testis was compared with the adjacent invasive GCT. All CIS lesions and invasive GCTs were aneuploid: CIS and seminomas were usual ly hyper t r ip lo id , and NSGCTs were usual ly hypot r ip- loid. The compar ison of the DI of CIS with its invasive GCT is summar ized in Table 1 (manuscr ipt in preparation).
The usua l ly lower DI of CIS in our material as compared with the DI repor ted by Mueller et al. [11, 18] is probably best expla ined by the fact that their pat ients were in the pre invas ive stage. Carcinoma in situ itself probably progresses through net loss of DNA.
Chromosome Numbers of Testicular Germ Cell Tumors and Carcinoma In Situ
Seminomas. A cytogenetic analysis of 14 pr imary seminomas was performed; 10 of the cases were pub l i shed previously [19]. Table 2 shows the modal number of normal copies of the different chromosomes and i(12p) of each case, as well as the modal chromosome counts. Four of the 14 seminomas lacked i(12p).
In all s tudied cases, chromosome counts of abnormal metaphases wi th in each tumor were rather homogeneous, which is in agreement with previous reports of Mart ineau [20] and Atkin [21], al though at variance with the findings of Delozier- Blanchet et al. [22]. In our experience, DNA flow cytometric graphs of seminomas are more homogeneous than those of NSGCTs, which lends further suppor t to our cytogenetic findings.
Most cytogenetic studies of TGCTs described a generally hyperd ip lo id to hyper- t r ip lo id chromosome complement with higher modal chromosome counts in semino- mas than in NSGCTs. Most of the reported seminomas and our cases show chromo- some numbers a round t r ip lo id and te t raploid values [19].
Tab
le
2 M
od
al
nu
mb
er
of
no
rmal
co
pie
s o
f ch
rom
oso
mes
an
d
i(1
2p
) p
er
case
Cas
e 1
2 3
4 5
6 7
8 9
10
11
12
13
1
4
15
16
1
7
18
1
9
20
2
1
22
X
Y
i(
12
p)
N*
1 3
4 2
4 3
5 5
4 4
6 3
7 2
8 4
5 3
4 5
5 5
2 2
3 3
3 2
3 4
3 1
3 2
3 2
3 3
2 2
2 2
4 5
3 4
3 5
3 3
4 4
5 3
3 2
5 1
4 8
5 2
3 4
4 5
4 4
5 3
4 3
5 6
6 5
3 3
5 3
4 5
4 3
4 4
5 7
5 3
3 4
2 2
3 4
5 2
3 1
3 2
3 4
2 1
1 3
3 2
6 3
3 3
2 2
2 4
4 3
2 2
3 2
4 3
3 2
1 3
3 5
3
7 3
2 3
2 2
3 4
4 3
3 2
3 2
3 4
3 3
2 3
3 4
5
8 1
2 2
2 2
3 3
2 2
3 1
4 1
2 2
2 2
1 3
3 2
1
9 3
3 1
2 2
3 4
4 2
2 1
3 1
2 4
2 3
2 3
3 4
3
10"
3 4
3 1
2 2
2 3
3 2
1 2
1 3
4 3
4 1
4 2
1 2
11
3 3
3 2
2 2
4 3
1 2
1 3
2 3.
5 4
3 3
2 3
3 2.
5 4
12
2 3
3 2
2 3
4 3
1 3
1 3
2 4
5 1
2 2
3 2.
5 4
.5
3 13
2
2 4
2 1.
5 3
3 4
1.5
2 1
2 1
1 4
3 2
2 3
3 3
5 2
2 4
11
2
3 2
1 2
68
7 4
2 1
-2
10
9
6 3
0 0
10
6
2 2
2 1
71
2 2
3 7
2
2 1
0 71
1 1
1 6
5
2 1
1 6
3
2 1
1 7
3
2.5
1.
5 1
68
2 1
0 7
1
2.5
2
1 2
65
1
4
3 1
3 2
2 3
3 3
2 2.
5 2
2.5
2 3.
5 3
3 1
2 3
3 4
4 2
1 0
To
tal
39
41
4
2
33
3
0.5
4
4
54
5
3
33
.5
39
.5
23
48
.5
24
4
6
55
41
33
2
9
48
4
6.5
5
4
49
.5
30
.5
17
.5
17
.5
66
N*
= m
od
al c
hro
mo
som
e n
um
ber
, st
ruct
ura
lly
ab
no
rmal
ch
rom
oso
mes
in
clu
ded
.
148 B. de Jong et al.
1.0_
0.6.
0.6.
0 .4_
0 .2_
M 0.0_
E A .02_ N
.0.4_
-0.6_
4).8_
-I .0
~::: iii~!
Mean ~And~'~Ted number of copies per chromosome (compared to 2)
Figure 2 Mean standardized number of the modal number of normal copies of chromosomes in 14 seminomas (as compared with two). For this calculation, the total number of normal chromosomes for each case was standardized to 46 to give every case an equal weight in terms of chromosomal counts, The mean number of sex chromosomes per case was multiplied by 2 to allow an easier comparison with the autosomes.
Since the first cytogenetic studies of seminomas, an apparent nonrandom gain or loss of certain chromosomes has been noticed (see Ref. 23 for review); we were able to confirm such an event in our sample. Table 2 shows the chromosomes that are usually over- or underrepresented.
Figure 2 shows the mean chromosome counts combined for all cases, after the total number of normal chromosomes was standardized to the arbitrary number of 46, Multiple comparison using the Newman-Keuls method showed that normal copies of chromosomes 11, 13, and 18 were underrepresented with respect to the normal copies of chromosomes 7, 8, 15, 21 and X (p ~ 0.01). Figure 3, deduced from the data shown in Figure 9, shows the (not standardized) numbers of normal plus structural abnormal copies of the different chromosomes in the group of the seminomas. Figure 4 shows a karyotype of one of the seminomas.
Nonseminomatous germ cell tumors. A cytogenetic analysis of 27 primary NSGCTs was performed; a full description of 14 of them has been published [241. Table 3 shows the modal number of normal copies of the different chromosomes and i(12p) for each case and the modal chromosome counts. Five of the 27 NSGCTs lacked the i(12p). Most reported NSGCTs and the ones in our study show hypotriploid
chromosome numbers [24]. Figure 5 shows the mean chromosome counts combined for all cases, after the total
number of normal chromosomes was standardized to the arbitrary number of 46.
Figure 5 Mean standardized number of the modal number of normal copies of chromosomes in 27 nonseminomatous germ cell tumors (NSGCTs} (as compared with 2). For this calculation, the total number of normal chromosomes for each case was standardized to 46 to give every case an equal weight in terms of chromosomal counts. The mean number of sex chromosomes for each case was multiplied by 2 to allow an easier comparison with the autosomes.
Figure 6 Mean number of short arms plus long arms for each specific chromosome in the group of nonseminomatous germ cell tumors.
Figure 7 Karyotype of one of the nonseminomatous germ (:ell tumors.
4
11
m W ,,,
19
5
12
t t
20
Mulitple comparison using the Newman-Keuls method showed that the normal copies of chromosomes 7, 8, 12, and X were overrepresented with respect to the normal copies of chromosomes 11 and 18 (p ~ 0.01). Figure 6, deduced from the data in Figure 9 show the numbers of normal plus structurally abnormal copies of the different chromosomes in the group of the NSGCTs. Figure 7 shows a karyotype of one of the NSGCTs.
Figure 8
I I
Karyotype of one of the cases of carcinoma ill situ of the testis.
s . s .........................................................................................................................................................................................
Average number of short arms plus long arms per specific chromosome in SEMINOMAS and NSGCTs
Figure 9 Average number of short arms plus long arms for each specific chromosome in the group of seminomas and nonseminomatous germ cell tumors. (Number of sex chromosomes multiplied by 2.)
Carcinoma in situ of the testis. We cytogenetically analyzed three cases of CIS of the testis; the three cases were published previously [25]. Owing to a very low yield of mitoses in all three cases, only one metaphase of substandard quality could be ana- lyzed. Table 4 shows the number of normal copies of the different chromosomes and i(12p) for each case.
All three cases were strongly aneuploid; two cases lacked i(12p). The number of chromosomes was in the peritriploid range. As compared with the corresponding invasive tumors, the karyotypes of CIS showed few structural chromosomal abnormal- ities. Figure 8 shows one of the karyotypes of CIS.
CYTOGENETIC AND PLOIDY STUDIES OF TESTICULAR GERM CELL TUMORS: REVIEW OF THE LITERATURE
Early cytogenetic studies of testicular GCTs described a generally hyperdiploid to hypertriploid chromosome complement with the modal chromosome number of semi- nomas higher (usually 60-69 chromosomes) than that of teratomas (50-59 chromo- somes) [20, 21, 26, 27], and combined tumors with intermedia.te modes [20]. Atkin [21] suggested the possibility that testicular tumors might arise from triploid rather than diploid or haploid cells. Wang et al. [28] reported cytogenetic evidence for premeiotic transformation o'f human testicular cancers. They observed simultaneous existence of X and Y chromosomes in the cells of 14 of 15 tumor cell lines. They concluded that their results were most compatible with a diploid origin of these tumors.
Recent studies [22, 29-35] confirmed the early observations about chromosome
Pathogenesis of Adult Testicular Germ Cell Tumors 155
numbers in the different testicular tumors. In 10 seminomas Atkin and Baker [33] found chromosome numbers ranging from 55 to 105 (mean, 82). Delozier-Blanchet et al. [22] found an average modal chromosome number of 69 for seminomas, of 54 for embryonal carcinoma, and of 59 for tumors of mixed histology. They found a significantly higher number of chromosomes in seminomas as compared with NSG- CTs. Gibas et al. [31] found four embryonal carcinomas with an average chromosome number of 67 and two immature teratomas with an average number of 59. In combined tumors (histologically recognizable as seminoma and teratoma), Martineau [20] found evidence that the two types of cells were chromosomally identical or at least had related karyotypes. Her studies, however, are difficult to interpret fully, since they were performed before the introduction of banding techniques. She found an under- representation of B and D group chromosomes and an overrepresentation of C group chromosomes. Berger et al. [36] and Haddad et al. [37] described a seminoma and an embryonal cell carcinoma occurring in different poles of the same testis, with both tumors containing related karyotypes. In six tumors, Berger et al. found an overrepre- sentation of the chromosomes 2, 7, 8, 12, 14-17, and 20. Atkin and Baker [29] found in their three seminomas an overrepresentation of chromosomes 12, and 19-22 and an underrepresentation of chromosomes 11 and 13. In a review of chromosomal abnormalities in TGCTs in both malignant teratomas and seminomas Sandberg [23] described a deficiency of group B and an excess of group C chromosomes. In semino- mas an excess of group F and a deficiency of chromosomes 17 and 18 have been also reported [see 23 for review]. After comparing the ratios of various pairs of groups, Sandberg [23] reported that pure tumors could be distinguished by the features of the various ratios of the different groups of chromosomes and that combined tumors appear to occupy an intermediate position in this respect.
The most common structural abnormality found in TGCTs is the isochromosome i(12p) [19, 22, 29-35]. This marker is characteristic of all histological varieties of germ cell tumors of the testis [22, 31]. Its occurrence in the various histological types of TGCTs points to their pathogenetic interrelationship. An i(12p) chromosome has also been found in two dysgerminomas [38, 39], one Mullerian mixed tumor [38], an ovarian yolk sac tumor [40], a mediastinal GCT [41], and in the metastasis of a malignant mixed gonadal stromal tumor of the testis [42]. An i(12p) has also been observed in Pallister-Killian syndrome [43]. Other, not consistent, structural chromo- somal abnormalities are observed in TGCTs. In 23 tumors, Delozier-Blanchet et al. [35] showed the chromosome regions most affected were 12p, 17q, lp and lq, 9q, 22q, 6q, and 7p. Abnormalities of chromosome 1 are often observed in TGCTs [22, 29, 31, 44-47] deletions of lp being the most frequent [22]. Parrington et al. [45-47] showed that cell lines established from testicular teratomas contain chromosome I rearrange- ments in addition to two apparently normal chromosomes 1. The intact chromosomes 1 appeared to be identical. In each of three seminomas, Atkin and Baker [29] found structural abnormalities of chromosome 1 and 12. The chromosome 1 changes in- volved duplication of lq and loss of lp.
Cytogenetic Comparison of Seminomas and Nonseminomatous Germ Cell Tumors (Unpublished Observations)
In essence, two models exist of the pathogenetic relationship between seminomas and NSGCTs. In the first model, the histogenesis of seminomas is assumed to diverge from that of the other TGCTs at an early stage. The neoplastic pathway of seminomas and NSGCTs is different, with no or only limited crossover. Consequently they show no or only a weak relationship. The second model suggests that seminomas and NSGCTs have a common origin with a single neoplastic pathway on which seminomas
156 B. de Jong et al.
~K
M E A N
N U M B E R
Average m~mher of short arms plus long arms per specific chromosome in SEMINOMAS and N ~ s
Figure 10 Average number of short arms plus long arms for each specific chromosome in the group of seminomas and nonseminomatous germ cell tumors.
are an in termedia te stage in deve lopment of NSGCTs. As a consequence, seminomas and NSGCTs show a strong relat ionship.
A compar ison of the cytogenetic data and p lo idy values of the different GCTs may shed some light on their relat ionship. For this comparat ive study, cytogenetic data from 14 pr imary seminomas and 27 pr imary NSGCTs were analyzed as follows. For each pat ient we de te rmined separately the modal number of short and long arms present for each chromosome, except the acrocentric chromosomes. Parts of chromo- somal arms involved in structural abnormali t ies were entered as whole arms if they represented 50% or more of the total arm length. Smaller segments were disregarded. The data obta ined were subjected to an analysis of variance to answer three main questions: Is the average number of short arms significantly different from the average number of long arms for specific chromosomes? Does the average number of short arms plus long arms per chromosome show significant differences between the two groups of tumors? Is there a characterist ic pat tern in the propor t ion of the different chromosomes present for each group of tumors? Data concerning the acrocentric chromosomes were analyzed similarly, taking into account the long arms only.
The average number of copies of i(12p) found in the two groups of tumors was analyzed by Kruskal l-Wall is analysis of variance [48] to test for the presence of differences be tween groups, fol lowed by comparisons between groups using the one- s ided Mann-Whi tney U test [48], wi th Bonferroni 's correction for mul t ip le compari - sons. The stat ist ical methods used and their respective results are descr ibed fully in the Append ix .
The average number of short arms plus long arms per specific chromosome in each group of tumors is shown in Figure 9. To provide a visual impress ion of the possible re la t ionship between seminomas and NSGCTs the data from Figure 9 are shown in graphs in Figures 10 and 11 (for all chromosomes and acrocentric chromosomes, respectively).
Pathogenesis of Adult Testicular Germ Cell Tumors 15 7
4.5 ._ l . . . . . . .m.+l ,k .q<~'T , ..................................................................................................................................................................................................... I i ,~MINOt~
+,.oi ....................... M E A 3.G N
N 3.0 U M B E zs+ R
2.0
1.5 1~1 1~ 211 ~
Average number of long arms of the acrocentric chromosomes per specific chromosome in SEMINOMAS and NSGCTs.
Figure 11 Average number of long arms of the acrocentric chromosomes for each specific chromosome in the group of seminomas and nonseminomatous germ cell tumors.
Comparison of the average number of copies of the different chromosomes in seminomas and NSGCTs showed a significant similarity in their relative proportions (Spearman rank correlation: 0.713, p < 0.001) (Fig. 10). We consider this finding (hardly conceivable in unrelated tumors) a convincing argument in favor of their common origin and relationship. Our data thus support the theory that seminomas and NSGCTs may have a single origin and a common neoplastic pathway.
In the two published cases of a combined tumor in which both components were separately karyotyped using banding techniques, [36, 37, 49] both components had clonal abnormalities in common, although in one case [49] the only structural abnor- mality in both components was i(12p). Martineau [20], studying unbanded chromo- somes, also found related karyotypes in the two components of combined tumors. In two macroscopically distinguishable components of a human TGCT, resembling a seminoma and an embryonal carcinoma, Walt et al. [50] reported two similar marker chromosomes, indicating their common origin. These karyotypic data support the model in which NSGCTs progress through a seminoma stage.
Origin of Aneuploidy in Seminomas and Carcinoma In Situ, or Both
Several hypotheses have been proposed regarding the way in which seminomas become aneuploid: successive nondisjunctions of a diploid cell, polyploidization, and cell fusion followed and/or preceded by chromosomal gain and/or loss [see 23 for review]. Because almost no cases of seminomas with less than 60 chromosomes have been reported successive random nondisjunctions in a diploid cell are not likely to be a mechanism of oncogenesis of seminomas. If they were, one would expect a higher frequency of (near) diploid seminomas and lower frequencies of seminomas with higher chromosome numbers. Because the opposite is observed, other modes of origin of aneuploidy should exist. The modal chromosome number of seminomas
158 B. de Jong et al.
shows a clustering around tri- and tetraploid numbers with a clear peak in the triploid range [19]. Mueller et al. [11, 18] found DI values of about 1.5-2.5 (mean, about 2) in testicular CIS cells, from which seminomas are presumably derived, in eight infertile men who had not yet developed invasive cancer. In patients with invasive cancer, we found DI values for CIS mostly in the same range as that of seminomas. Therefore, the clustering of modal chromosome numbers of seminomas around the triploid range may be explained by chromosome nondisjunctions in a tri- to pentaploid cell, leading mostly to net loss of chromosomes. The polyploid cell from which seminomas derive may result either from mitotic or meiotic errors leading to tri- or tetraploidization or from fusion between a haploid with a diploid cell or fusion of cells with other ploidy values.
Most ovarian and extragonadal GCTs (including teratomas) are (near) diploid [see 51 for review]. Polyploidization is a rather unique feature for TGCTs, shared by dysgerminomas of the ovary [52] and mediastinal GCTs [53]. This propensity for polyploidization appears to be related to gonocyte derivation [and presence of i(12p)?] of these tumors. Gonocytes from which abnormal premalignant intratubular cells are derived may be particularly prone to polyploidization because they combine high mitotic activity and the phenomenon of bridge formation. In particular, a defective mechanism of bridge formation has been implicated in the pathogenesis of GCTs [54]. Cell fusion may be facilitated by bridge formation.
In vivo hybridization or polyploidization may play a significant role in oncogenesis and neoplastic progression [21, 23, 55-57]. The existence of aneuploidy might provide a basis for continuing nondisjunction and enhance the genetic instability of neoplastic cells. Polyploidization as an early step in tumor progression followed by chromosome loss is well documented for bladder and prostate cancer [58] and has also been demonstrated in an experimental tumor model [59]. Recently, several features of a tumor progression model, starting with tetraploidization and followed by loss of chromosomes, were described [60].
Tumor Progression of Testicular Germ Cell Tumors
Most often, progression of a malignant tumor is the result of clonal evolution of a tumor cell population, characterized by increasing aneuploidy and genetic instability of tumor cells, increasing proliferative potential, decreasing capacity of differentia- tion, and greater malignant potential [57, 61-71].
Contrary to the tumor progression model originally proposed and extended by Nowell [62-64] in which the clonal evolution of a tumor cell population progresses from diploid to hyperdiploid or highly aneuploid chromosome numbers, in TGCTs tumor progression may progress from high to lower numbers of chromosomes. This would mean that in addition to gain of chromosomes or parts of chromosomes and development of structural chromosomal abnormalities, loss of chromosomes or chro- mosomal material should be a very early fundamental and important feature of tumor progression of TGCTs. The tumor progression model of TGCTs will probably consist of the following components, although not necessarily in the order shown: (1) aneu- ploidy owing to cell fusion or polyploidization; (2) genetic instability due to aneu- ploidy [61, 72]; (3) development of structural chromosomal abnormalities, especially i(12p) formation, leading to oncogene deregulation [57, 61-68, 73-75]; and (4) gain or retention of chromosomes or chromosomal material leading to a selective growth advantage, loss of chromosomes or chromosomal material where the loss will repre- sent a selective growth advantage (e.g., by loss of tumor suppression [69, 70, 76-83], or loss of the ability of terminal differentiation [69, 78].
TGCTs usually are highly aneuplold. The i(12p) chromosome is observed in about 80% of all TGCTs (Tables 2 and 3). This frequent occurrence of i(12p) indicates that it plays an important role in the oncogenesis of TGCTs, as does aneuploidization.
Pathogenesis of Adult Testicular Germ Cell Tumors 159
That all three cases of CIS were strongly aneuploid and that two cases lacked i(12p) [although in these two cases the invasive cancer also lacked the i(12p)] may indicate either that formation of this marker is not a necessary event in carcinogenesis or that it is proceeded by polyploidization [25]. The latter hypothesis is in agreement with the finding that chromosome 12q heterozygosity is retained in i(12p)-positive TGCTs [84].
The progression of TGCTs probably proceeds from high to lower numbers of chromosomes and is therefore accompanied by a net loss of chromosomal material. This decrease will be the end result of loss of specific chromosomes, gain of some other chromosomes (or part of chromosomes), and development of structural abnormalities.
Loss of certain chromosomes or (loss of heterozygosity for) some chromosomal regions is important for development of malignancy, presumably because of loss of genes with tumor-suppressing and differentiation-regulating properties [77-83, 85-91]. If los~ of chromosomes in TGCTs is related to loss of genes crucial for normal cell differentiation, different chromosomes should be underrepresented in NSGCTs as compared with seminomas. Because both are germ cell tumors, however, it is not surprising that some chromosomes are underrepresented in both subtypes.
Both in seminomas and NSGCTs, specific chromosomes were consistently under- represented (e.g., 11, 13, 18, and Y), whereas other chromosomes were consistently overrepresented (e.g., 7, 8, 12, and X). The chromosomes consistently underrepre- sented may contain genes important for normal germ cell differentiation or have tumor-suppressing properties or both. Chromosomes consistently overrepresented may contain genes responsible for a more malignant development. The average num- ber of copies of i(12p) is significantly higher in NSGCTs than in seminomas.
As compared with seminomas, some acrocentric chromosomes (especially chromo- somes 15 and 22) have significantly fewer copies in NSGCTs. Thus, loss of these chromosomes may be crucial for a seminoma stage cell to evolve into a NSGCT, possibly because of loss of genes important for germ cell differentiation.
In two different samples of NSGCTs, we found an identical over- and underrepre- sentation of specific chromosomes. This supports the view of a functional relationship between gain and loss of specific chromosomes and the process of oncogenesis and tumor progression [92].
Loss of heterozygosity might be important for development of malignancy. The chromosomes most often underrepresented are the most likely candidates to show gene loss. Loss of heterozygosity in TGCTs has been demonstrated for the chromosome arms lp, lq, 5q, 7q, and 11q [93], 3p and 11p [94], and 11p [95]. Loss of heterozygosity of chromosomes 5 and 11 is in agreement with our cytogenetic findings; loss of heterozygosity of the chromosomes 1 and 7 is not. Loss of heterozygosity will not always be accompanied by visible loss of chromosomes, however. Moreover, progres- sion of TGCTs is a combination of events [loss of chromosomes, loss of heterozygosity, gain of chromosomes, i(12p) formation, and development of other structural abnor- malities]; in individual patients, the combinations may be very different. Alteration and/or activation of the c-myc oncogene [96], of ras oncogenes [97], or of homeobox- containing genes [98] ,may play a role in the oncogenesis and progression of TGCTs.
PATHOGENETIC MODEL: CONCLUSION
Our data on the cytogenetics and ploidy of seminomas, combined tumors, and NSGCTs lend support to the model of pathogenesis of seminomas and NSGCTs proposed by Ewing and Friedman [16, 17], in which all TGCTs (with the possible exception of spermatocytic seminoma and IYST) have a single origin and neoplastic pathway, with seminomas representing "an intermediate stage in development of NSGCT components, as opposed to the model suggested by Pierce and Abell [14] in which seminomas and NSGCTs develop separately.
160 B. de Jong et al.
2
i i
1,5 . . . . . . . . . . . . . . . . I . . . . . . . . . . . L __ _ '_4t i4 i~ l t . . . . . . . . . . . . . . . . . . . . . . ~ i l l , i . . . . . . . . . . l i l L . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . =='IF ""===II~
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approx, age of clinical presentation
young to adolescent age adult age
Figure 12 Schematic representation of a model of tumor progression in germ cell tumors of the adult testis. Supposedly, an early event is polyploidization of a dysplastic germ cell precursor, resulting in carcinoma in situ with a DNA index of about 2. Initial net loss of DNA (chromosomes or parts of chromosomes endowed with tumor-suppression properties), leads to invasive semi- noma (solid line), a tumor type through which all other types progress. Rapid progression through the seminoma stage by further loss of DNA leads to nonseminoma (dotted line). Slower progression through the seminoma stage may result in combined tumor. Seminomas, being the least aggressive, become clinically manifest at older age than the more aggressive nonseminomas. Combined tumors have an age of c]inical presentation in between the two [13].
Our results fit with a pathogenetic model shown in Figure 12. Carcinogenesis probably starts during intrauterine life [99-101]. An early event might be polyploid- ization of a dysplastic germ cell precursor, resulting in CIS with a hypertriploid to peritetraploid DNA content. The mean DI of CIS cells was between 1.5 and 2.5 in eight infertile men who had not yet developed an invasive GCT [11, 18]. Another early event is formation of i(12p). In i(12p)-negative TGCTs [102], other chromosomal events (e.g., other structural abnormalities of chromosome 12) may substitute for i(12p) formation.
Tumor progression in TGCTs conceivably is the result of a combinat ion of chromo- somal events [e.g., loss of chromosome 5, 11, 13, and 18; gain of chromosomes X, 7, 8, and 12; i(12p) multiplication; development of structural abnormalities leading to oncogene deregulation; loss of heterozygosity; oncogene activation by point muta- tions] causing a net loss of DNA. The first stage that may be reached in this progression is a seminoma stage. In about half of the cases, the neoplastic pathway may end here and proceed along the same line of differentiation, resulting in invasive seminoma, a tumor in which the cells resemble gonocytes, as do CIS cells [5]. They have similar ultrastructural features [103] and both express PLAP [4]. The DI values of CIS and seminomas are comparable and in general higher than those of NSGCTs. Seminomas, which are less aggressive than NSGCTs and probably earlier in the clonal evolution [62] will become clinically manifest at older age, with most patients in stage I [104].
Pathogenesis of Adult Testicular Germ Cell Tumors 161
In the other half of the cases, the neoplastic pathway may evolve through a usually subclinical, probably in situ, seminoma stage by further net loss of chromosomes [again a combination of chromosomal events: loss of chromosomes 15 and 22, gain of more copies of the i(12p)], leading to a NSGCT, a more advanced cancer in terms of tumor evolution, which has lost the capability of gonocytic differentiation. On the average, the tumor is more aggressive than seminoma, and thus becomes manifest at a younger age. Patients with combined tumors present at an age in between that of seminoma and NSGCT patients [6, 105]. In our study, the median age of patients presenting with combined tumors is significantly higher than that of NSGCT patients and significantly lower than that of patients presenting with a seminoma [13]. A striking finding is that the median DI of the seminoma and the NSGCT components in combined tumors is similar to the DI of the pure seminoma and NSGCT counter- parts. In most of the cases in which the two components could be measured separately, the seminoma component had the higher DI. This observation is compatible with the hypothesis that the NSGCT component has evolved from the seminoma component through net loss of chromosomes.
Progression of TGCTs probably proceeds only when certain critical chromosomal events or combinations of events occur. That these critical events may occur early or later in the process of random chromosomal loss, gain, and alterations may account for the wide variation in DI of TGCTs.
The model does not imply that all patients with a NSGCT do or did show a clinically manifest seminoma stage. Progression may rapidly pass through a subclini- cal, probably in situ, seminoma stage when the appropriate chromosomal events leading to a NSGCT occur. Direct progression of a seminoma to a NSGCT in a meta- static lesion may have occurred in patients who have testicular seminoma and metas tases with nonseminomatous elements [see 106 for review]. Nonseminomatous GCT elements have been found in up to one third of patients dying of seminoma [107].
It is evidently more difficult to fit IYST into the proposed model. Of 10 tumors, three are diploid; apparently polyploidization is not obligate. When polydiploidization has occurred, the DI of the tumors remains close to 2 (1.91), in keeping with a short period of tumor evolution in young patients. The relatively low malignant potential of the tumors is reflected in the high proportion of patients who are in stage I [108]. The entirely different histological composition of TGCTs of infants as compared with TGCTs in adults [109, 110] may be explained by epigenetic factors in the infantile testis. The pathogenesis of TGCTs of infants also may be fundamentally different from those of adults, because CIS could not be demonstrated in the vicinity of IYST [10]. On the other hand, it is found in a very high proportion of testes containing GCT in adults [10, 111], with the exception of spermatocytic seminoma [11]. Additional evidence for a different pathogenesis may be the occurrence of diploid tumors, and the absence of the i(12p) marker chromosome in the only IYST that we have karyo- typed [112]. This marker was present in more than 80% of testicular GCTs in the adults we karyotyped. The pathogenesis of spermatocytic seminoma also may be different. These tumors are negative for PLAP [4] and are not composed of gonocytes but of more mature germ cell precursors.
Our data on ploidy and cytogenetics of seminoma and NSGCTs fit nicely into the pathogenetic model in which all TGCTs of adults progress through a seminoma stage. If this model is correct, it will offer a system to study germ cell activation: the switch from a highly specialized germ cell to a pluripotent embryonal cell. A careful comparative cytogenetic study of the two tumor types may show which chromosomes harbor genes important for differentiation, which chromosomal events are important for oncogenesis and tumor progression, and which oncogenetic steps are common or different for different GCTs.
1 6 2 B. de Jong et el.
T a b l e 5 A n a l y s i s of v a r i a n c e for t he n o n a c r o c e n t r i c c h r o m o s o m e s
Source Sum of squares df Mean-square F Ratio p Value
Tumor type 38.339 1 38.339 34.250 0.000 p or q 5.358 1 5.358 4.787 0.029 Chromosomes 704.486 17 41.440 37.021 0.000 Tumor type ~ p or q 0.635 1 0.635 0.567 0.452 Tumor type ° Chromosomes 45.746 17 2.691 2.404 0.001 p or q~ Chromosomes 145.406 17 8.553 7.641 0.000 p or q~ Tumor type ° Chromosomes 29.398 17 1.729 1.545 0.072 Error 1,571.614 1,404 1.119
Abbreviation: dr: degrees of freedom.
°Interaction.
APPENDIX
Statistical Analysis
T h e n u m b e r of c o p i e s of the p a n d t he q a r m for the n o n a c r o c e n t r i c c h r o m o s o m e s in two t y p e s of t e s t i s t u m o r s , s e m i n o m a s , a n d NSGCTs was t a k e n as the d e p e n d e n t va r i ab le . G r o u p i n g fac tors w e r e t he t y p e of t h e t u m o r , t he p or q o b s e r v a t i o n , a n d the c h r o m o s o m e n u m b e r . T h e p or q o b s e r v a t i o n s are in m o s t cases d e p e n d e n t , b e c a u s e one i n t a c t c h r o m o s o m e l eads to a c o u n t for the p a n d for t he q part . T h e da ta for the n o n a c r o c e n t r i c c h r o m o s o m e s we re a n a l y z e d as a 2 × 2 x 18 fac tor ia l des ign , w i t h t he n u m b e r of o b s e r v a t i o n s d e p e n d e n t o n the t ype of t u m o r . T h e a c r o c e n t r i c c h r o m o - s o m e s w e r e a n a l y z e d in a 2 x 2 x 6 fac tor ia l d e s i g n ( t umor s x c h r o m o s o m e s ) . T h e da ta c a n be d e s c r i b e d w i t h t h i s m o d e l , w h i c h e x p l a i n s 41 .3% of the to ta l va r i ance . T h e m o d e l is: n u m b e r of c o p i e s o b s e r v e d = c o n s t a n t + c h r o m o s o m e - d e p e n d e n t v a l u e + t u m o r - t y p e d e p e n d e n t v a l u e + v a l u e d e p e n d e n t o n p or q a r m (for t h e n o n a c r o c e n t r i c c h r o m o s o m e s ) + all i n t e r ac t i ons .
T a b l e s 5 a n d 6 s h o w t he a n a l y s i s of v a r i a n c e of th i s m o d e l , as c o m p u t e d by t he S Y S T A T [113] p rog ram. Mos t of t he v a r i a n c e is e x p l a i n e d by d i f f e r ences in c h r o m o s o m e c o u n t s pe r c h r o m o s o m e . T h e i n t e r a c t i o n b e t w e e n the p or q c l a s s i f i ca t ion a n d t he c h r o m o s o m e c l a s s i f i ca t ion is e n t i r e l y d u e to the fact t ha t 12p a r m s are ve ry
T a b l e 8 A n a l y s i s of v a r i a n c e for t he a c r o c e n t r i c c h r o m o s o m e s
Source Sum of squares df Mean-square F Ratio p Value
number Tumor type ° Chromosomes 32.553 5 6.511 6.612 0.000 Error 230.403 234 0.985
Abbreviation: df, degrees of freedom.
°Interaction chromosome/tumor type.
Pathogenesis of Adult Testicnlar Germ Cell Tumors 163
frequent in comparison to 12q arms. The differences dependent on the chromosome number (main effect) is shown in Fig. 10. Chromosomes 7 and 12 are present in higher numbers than the others. The significant interaction between chromosome number and tumor type is mainly explained by noting that the ratio of the number of copies of chromosome 12 in comparison to chromosome 7 depends strongly on the tumor type. In quantitative terms, this interaction is the least important effect, as the F ratio is only 2.404 as compared with more than 7.641 for the other significant effects.
For the acrocentric chromosomes, the interaction between chromosomes and tumor type is due to the relatively higher number of copies of chromosomes 14, 15, 21, and 22, in comparison to chromosomes 13 and Y for the seminomas in contrast with that in the NSGCTs.
This work was supported by Grants No. GUKC 84-8 and 88-10 from the Netherlands Cancer Foundation (KWF) and by the Jan Kornelis de Cock Stichting. The authors thank Mentje Dijkstra for typing the manuscript and Rene Kraaijenbrink for making the graphs.
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