Histogenetic Analysis of Ovarian Germ Cell Tumors by DNA Fingerprinting

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[CANCER RESEARCH 52, 6823-6826, December 15, 1992] Histogenetic Analysis of Ovarian Germ Cell Tumors by DNA Fingerprinting

Masaki Inoue, 1 Masami Fujita, Chihiro Azuma, Fumitaka Saji, and Osamu Tanizawa Department of Obstetrics and Gynecology, Osaka University Medical School, Osaka 553, Japan

ABSTRACT the other hand, Carritt et al. (14) observed a heterozygous pat- tern of chromosomal heteromorphism in four ovarian terato- The histogenesis of ovarian germ cell tumors (11 mature teratomas, mas and proposed that ovarian teratomas might originate from three malignant transformations of mature teratomas, two immature a germ cell prior to the first meiosis. This speculation was teratomas, and four dysgerminomas) was investigated genetically using minisatellite DNA probes 33.15 and 33.6 for person-specific restriction further supported by Zeuthen et al. (15) who observed centro- fragment length polymorphism (DNA fingerprint) analysis. The DNA metric heterozygosity in one ovarian teratocarcinoma cell line. fingerprints of six ovarian teratomas were identical with those of mono- Recently, however, some investigators (16, 17) observed both nuclear cells from each host, while some polymorphic bands observed in heterozygosity and homozygosity of chromosome and enzyme the host mononuclear cells were lost in the DNA fingerprints of the markers in several ovarian tumors and postulated that ovar- other five cases. The cases of malignant transformation of mature ter- ian teratomas arise from germ cells in a number of different atoma and immature teratoma showed that some polymorphic bands of ways. Thus, the pathogenesis of ovarian teratomas has not DNA fingerprints from the host mononuclear cells were absent in the been completely clarified by conventional cytogenetic methods tumor tissues. In four cases with dysgerminomas, the DNA fingerprints and remains to be investigated by more sensitive and specific of tumors were completely identical with those of the respective host techniques. mononuclear cells. The present results suggest that mature cystic teratomas of the ovary arise from germ cells arrested at various stages of Recent advances in molecular technology permit the charac- meiosis, while immature teratomas are derived from postmeiotic germ terization of genetic variations at the DNA level. One highly cells. Malignant transformation may occur exclusively in the mature sensitive and discriminating method of analysis is based upon teratomas arising from postmeiotic germ cells. Dysgerminomas develop DNA polymorphisms, which can be detected as RFLPs 2 by a from premeiotic oogonia (primodial germ cells). Thus, DNA finger- probe based on a tandem-repeat of the core sequence (18). One prints are a useful and sensitive tool for identifying the pathogenesis of such probe, the minisatellite DNA probe described by Jeffreys germ cell tumors. et al. (19, 20) can detect many regions of great variability within the human genome and can provide an individual-specific DNA INTRODUCTION "fingerprint." Germ cell tumors embrace morphologically heterogeneic We have reported a preliminary observation on the histoge- neoplasms considered to be originated from germ cells of the nesis of ovarian teratomas using a minisatellite DNA probe, gonad and are the most common ovarian tumors in women 33.15, and suggested that mature cystic teratomas of the ovary during the second and third decades of life, accounting for arise from germ cells before Meiosis I, while immature terato- approximately 20% of all ovarian tumors (1). However, the mas are derived from germ cells after Meiosis I (21). In the histogenesis of these tumors is unclear and has been a matter of present study, a larger number of cystic teratomas, their malig- speculation and dispute for decades. Much of the early work on nant transformations, immature teratomas, and dysgermino- the histogenesis of ovarian germ cell tumors was done by Tei- mas of the ovary were analyzed to determine the origin of such lum (2, 3), who proposed that such neoplasias originate from types of ovarian germ cell tumors using minisatellite DNA primodial germ cells and could give rise to either dysgermi- probes 33.15 and 33.6. noma or tumor of totipotential cells; the latter could then dif- ferentiate into embryonal carcinoma, which in turn could form MATERIALS AND METHODS neoplasms of extraembryonic structures (yolk sac tumors and choriocarcinomas), or embryonic structures (teratomas). These Patients. Samples were obtained from 20 patients with ovarian tumors who underwent laparotomy at the Department of Obstetrics and views based on histopathological stu'dies were further supported Gynecology, Osaka University Medical School. Histological diagnosis by the embryological studies of Witschi (4) and Gilhnan (5) and was carried out according to the WHO histological typing system (22). later by the experimental work of Stevens (6) and Pierce et al. The clinicopathological data are listed in Table 1. (7, 8) on germ cell tumors in rodents. We removed a small piece of tissue from the center of each tumor, In recent times, two main theories for the histogenesis of taking care not to include any material from the ovarian capsule, and teratomas have been proposed: an origin from segregated blas- washed it repeatedly in cold phosphate-buffered saline, pH 7.2, to ex- tomeres at an early stage of embryonic development and an clude contamination by the blood of the host. Mononuclear cells origin from a parthenogenetic primodial germ cell. The latter were also obtained from the peripheral blood of the patients for DNA theory has been steadly gaining support in clinicopathological analysis. studies, animal experiments, and cytogenetic studies and is now DNA Analysis. High-molecular-weight DNA was extracted from the tumor tissues and mononuclear cells of the patients as described generally accepted (9, 10). Linder and coworkers (11-13) dem- elsewhere (23). Four #g of each DNA sample were digested with endo- onstrated that benign ovarian teratomas arise from a single nuclease Hinfl at 37~ for 3 h, electrophoresed on 0.7% agalose gel, germ cell after the first meiotic division using both cytogenetic and then transferred by blotting to nitrocellulose filters according to the techniques and electrophoretic variants of enzyme markers. On method of Southern (24). After baking the filters under a vacuum at 80~ for 3 h, a 32p-labeled minisatellite DNA probe was hybridized at Received 5/21/92; accepted 9/30/92. 62"C for 12 h in Denhardt's solution, 1 M NaC1, 50 mM Tris-HCl The costs of publication of this article were defrayed in part by the payment of (pH 7.4), 0.1% sodium dodecyl sulfate, 10 mM EDTA, and 0.1 mg/ml page charges. This article must therefore be hereby marked advertisement in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact. of denatured sonicated salmon sperm DNA. The filters were then i To whom requests for reprints should be addressed, at Department of Obstet- rics and Gynecology, Osaka University Medical School, 1-1-50, Fukushima, Fuku- shima-ku, Osaka 553, Japan. 2 The abbreviation used is: RFLP, restriction fragment length polymorphism. 6823 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1992 American Association for Cancer Research. DNA FINGERPRINT OF OVARIAN GERM CELL TUMORS

Table 1 Clinicopathological findings and DNA fingerprintings of germ cell polymorphic bands, constituting person-specific RFLPs called tumors of the ovary DNA fingerprints, while fragments smaller than 4 kilobases do DNA not show person-specific polymorphisms (20). In the six cases fingerprinting Age (pre- and (Cases 1 to 6) of 11 mature teratomas (dermoid cysts), each Case (yr) P/G a Diagnosis post-Meiosis 1) DNA fingerprint was identical to that of the mononuclear cells 1 32 1/3 Mature teratoma Pre from the host. In the other five cases, some polymorphic bands 2 25 0/0 Mature teratoma Pre observed in the host were deleted from the DNA fingerprints of 3 46 2/3 Mature teratoma Pre 4 68 4/4 Mature teratoma Pre the tumor tissue. It is very interesting that all three cases of 5 33 2/3 Mature teratoma Pre malignant transformation from a dermoid cyst showed fewer 6 17 0/0 Mature teratoma Pre 7 26 l/l Mature teratoma Post polymorphic bands than the host mononuclear cells. Two im- 8 20 0/0 Mature teratoma Post mature teratomas also showed that some polymorphic bands 9 28 2/2 Mature teratoma Post observed in the host were deleted from the DNA fingerprints of 10 21 0/0 Mature teratoma Post 11 28 2/3 Mature teratoma Post the tumor tissue. In all four dysgerminomas, the RFLP band patterns of the tumor tissues were identical with those of each 12 70 5/6 Malignanttransformation Post of a mature teratoma host. 13 56 2/4 Malignanttransformation Post Table 1 summarizes the conclusions based on the results of of a mature teratoma the present study using minisatellite DNA probes 33.15 and 14 64 6/3 Malignanttransformation Post of a mature teratoma 33.6. In the course of development, the primitive germ cells undergo two special divisions, Meiosis ! and Meiosis II. Just 15 18 0/0 Immatureteratoma Post before Meiosis I, the primitive germ cells replicate their DNA 16 21 0/0 Immatureteratoma Post and contain 46 chromosomes, each of which is a double struc- 17 19 0/0 Dysgerminoma Pre ture. After Meiosis I, the number of chromosomes is reduced to 18 16 0/0 Dysgerminoma Pre half the number of a somatic cell (23 double-structured chro- 19 24 0/0 Dysgerminoma Pre 20 22 0/0 Dysgerminoma Pre mosomes), while the amount of DNA equals that of a somatic a P/G, parity (the number of pregnanciesreaching viability)/gravida (the number cell. In Meiosis II, the 23 double-structured chromosomes are of pregnancies irrespective of their outcome). divided to single structures, at which point the amount of DNA is half that of a somatic cell. Consequently, a finding that some washed once at room temperature in 2• sodium chloride-sodium polymorphic bands are deleted in the tumor DNA may suggest citrate, followed by two washes at 65~ in I x sodium chloride-sodium that the tumor arose from a postmeiotic stage germ cell. Con- citrate containing 0.1% sodium dodecyl sulfate for 30 min, and they versely, a finding that the tumor and the host have the same were autoradiographed using Kodak XRP-I film (Kodak, Rochester, band patterns could indicate that the tumor arose from a pre- NY) at -80~ for 48 h. meiotic germ cell. Thus, the present results suggest that mature The minisatellite DNA probes 33.15 and 33.6 used for the present cystic teratomas of the ovary arise from germ cells arrested at RLFPs analysis were kindly provided by Alfred J. Jeffreys (19, 20). The probes were labeled with [32P]dCTP by the modified nick translation various stages of meiosis, while immature teratomas are derived method to a specific activity of 1.8 x 109 cpm/ug (25). from germ cells after Meiosis I. The malignant transformation may take place in the mature teratomas derived from germ cells after Meiosis I. Dysgerminomas arise from premeiotic germ RESULTS cells. Fig. 1 shows the representative profiles of the RFLP band patterns detected by minisatellite DNA probes 33.15 and 33.6 DISCUSSION in six sets of the ovarian germ cell tumor tissues and the peripheral mononuclear cells of the respective host. In the 4- Early genetic studies of germ cell tumors relied heavily on to 20-kilobase region, each patient displays about 10 to 20 analysis of sex chromatin (Barr bodies) and were complicated

Case 1 Case 8 Case 12 Case 15 Case 19

(33.15) (33.6) (33.15) (33.6) (33.15) (33.6) (33.15) (33.6) (33.15) (33.6) Fi$. 1. The representative band patterns of TH TH TH TH TH TH TH TH TH TH restriction fragment length polymorphismsof DNA of ovarian germ cell tumors in tumor tissues (T) and the mononuclear ceils of the 20 kb -mm,-- respective hosts (H) obtained with the 33.15 15kb,-~; and 33.6 minisatellite probes. The case num- bers correspond to those in Table 1. The DNA fingerprint of mature teratoma is identical to lOkb ,-e,. that of the host mononuclear cells in Case 1, while some polymorphic bands (arrows) are deleted from the tumor tissues in Case 8. In Case 12 (malignant transformation of a mature teratoma) and Case 15 (immature teratoma), some polymorphic bands (arrows) detected in the host were not observed in the tumor tissues. In Case 19 (dysgerminoma),the fingerprintings of tumor tissues are identical to those of the respectivehost monoclearcells. 4kb,-~ kb, kilobases.

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1992 American Association for Cancer Research. DNA FINGERPRINT OF OVARIAN GERM CELL TUMORS by inaccurate observations and an inability to predict the geno- chromosome analysis (29-31). On the other hand, two imma- type from the nuclear sex (10). Karyotypic analysis of benign ture teratomas showed fewer bands of polymorphic fragments cystic teratomas of the ovary soon revealed a diploid 46XX than those of their host mononuclear cells, suggesting that im- genotype in most benign lesions (26, 27). Using the analysis of mature teratomas arise from postmeiotic germ cells. A few new electrophoretic variants of enzyme markers, Linder and Power bands seem to have appeared in tumor tissue of Case 15 when (11, 12) demonstrated some heterozygous host alleles distant compared with the host mononuclear cells. However, these from the centromere in some ovarian teratomas, although most bands are also observed in the lane of the host, even though they teratomas arising in 31 heterozygous hosts were homozygous. are very weak. Such a difference in the intensity of bands be- Chr0m0s0me-banding heteromorphisms located near the cen- tween tumor and host DNA samples could be caused by a tromere have also been shown tO be homozygous in most ova- difference in the degree of DNA methylation, which could rian teratomas (13, 28). Therefore, it has been speculated that affect the efficacy of Hinfl digestion. As another possible ex- teratomas are derived from post-Meiosis I cells and that het- planation, there may be rearrangement or mutation of tumor erozygous teratomas arise from cross-over between the cen- DNA in the chromosomal regions adjacent to the hypervariable tromere and the relevant locus (11-13, 28). tandem-repeat intron arrays which are detected by these probes Although most ovarian benign teratomas are postmeiotic, (19,34). several cases with heterozygous centrometric banding hetero- It is interesting that the bandings of DNA fingerprintings morphisms have been reported, suggesting that some teratomas from tumor tissues were fewer in number than those of the may arise from a premeiotic germ cell. Parrington et al. (29) respective host in all three cases with malignant transformation found that 8 of 12 teratomas arising in 10 patients displayed of a mature teratoma, indicating that malignant transformation heterozygous centrometric heteromorphism identical to that of may occur exclusively in mature teratomas arising from post- the host. Six of these 8 teratomas developed in 3 patients with meiotic germ cells. Since such tumors arising from the post- multiple teratomatosis. Nomura et al. (30) also applied chro- meiotic germ cells have fewer chromosomes than the host, they mosome banding, human leukocyte antigen determination, and may easily and often transform to a malignant phenotype, and enzyme analysis to 16 teratomas from 4 patients and found that it may be that during that process the deletion of a tumor 3 teratomas in 2 patients had heterozygous centrometric mark- suppressor gene takes place. Actually, we have detected the ers identical to those of the host. A triploid mature ovarian overexpression and point-mutation of the tumor suppressor teratoma arising in a diploid host was recently described, sug- gene, p53, in such tumors. 3 gesting another mechanism by which a haploid polar body may Histogenetic analysis of dysgerminoma has been less com- be fused with a diploid ovum (31). Thus, the results obtained by mon, compared to teratomas. The morphological similarity be- chromosome analysis have supported the speculation that between dysgerminoma cells and germ cells of the developing nign ovarian teratomas arise from germ cells in a number of ovary strongly suggests that these tumor cells are of germ cell different ways. origin. Although some investigators (35) reported that the In the present study, we introduced a newly developed cells of some dysgerminomas have sex chromatin bodies, method of DNA fingerprinting for histogenetic analysis of ova- Asadourian and Taylor (36) found that the tumor cells of 22 rian teratomas. The probes 33.15 and 33.6 used in the present dysgerminomas were sex-chromatin negative, while the stromal study can simultaneously detect highly variable regions widely cells were sex-chromatin positive. They also showed that the dispersed in the human genome and can provide individual- DNA content in the nuclei of dysgerminoma cells is approxi- specific RFLPs called DNA fingerprints (18, 19). In view of the mately double the content of the control somatic cells. All of fact that these polymorphism fragments are inherited from host these findings point toward an origin from primodial germ cells cells in a Mendelian fashion, DNA fingerprint analysis is very at the beginning of the first meiotic division. The present study useful in many genetic investigations, such as zygosity determi- using DNA fingerprints supports such a view. The recently nation in multiple pregnancy, mapping of human linkage developed method using the specific probes, 33.15 and/or 33.6, groups, indirect determinations of genetic disease loci, and de- definitely provides greater accuracy than the conventional ge- tection of meiotic recombinations (25, 32). Along these lines, netic tests and offers a useful and sensitive tool for the cytoge- we previously reported a new application of minisatellite DNA netic analysis of germ cell tumors. probe 33.15 to the genetic analysis of complete hydatidiform moles and demonstrated the value of this probe for the verifi- ACKNOWLEDGMENTS cation of androgenesis as the cause of complete moles (33). The germ cells are well known to undergo two special divi- The authors wish to thank Masahiko Hori and Kiyofumi Kamiyama of Teijin Bio Laboratories, Inc., Tokyo, Japan, for their excellent tech- sions, Meiosis I and Meiosis II, by which the number of chro- nical assistance. mosomes and then the amount of DNA are reduced to half those of a somatic cell, respectively. Deletion of several poly- REFERENCES morphic bands in a tumor tissue could suggest that the tumor arose from a postmeiotic stage germ cell. As shown in Fig. 1, 1. Scully, R. E. Teratomas. In: Atlas of Tumor Pathology, Fascicle 16, pp. 246-252. Washington, DC: Armed Forces Institute of Pathology, 1979. the DNA fingerprints of six ovarian teratoma tissue specimens 2. Teilum, G. Endodermal sinus tumor of the ovary and testis. Comparative were identical with those of the respective host mononuclear morphogenesis of the so-called mesonephroma (Schiller) and extra-embryonic (yolk sac ailantoic) structures of the rat placenta. 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DNA FINGERPRINT OF OVARIAN GERM CELL TUMORS

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Masaki Inoue, Masami Fujita, Chihiro Azuma, et al.

Cancer Res 1992;52:6823-6826.

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