Histogenetic Analysis of Ovarian Germ Cell Tumors by DNA Fingerprinting

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