Malignant Transformation of Ovarian Epithelium Robert C. Bast, Jr.,* Ian Jacobs, Andrew Berehuek Ovarian cancer is the leading cause of gynecologic cancer deaths in the United States. In contrast to cervical cancer, where etiologic agents and early neoplastic lesions have been clearly defined, the pathogenesis and natural history of ovarian cancer remain obscure. More than 90% of ovarian cancers in adults are thought to arise from a single layer of epithelial cells that cover the ovarian surface or line inclusion cysts. Following ovulation, the ovarian surface epithelium proliferates to repair the defects produced by rupture of mature follicles. Epidemiologic data suggest that the interruption of ovulation by pregnancy, lactation, or oral contraceptive medications substantially reduces the risk of developing ovarian cancer. Suppression of ovulation may decrease opportunities for tumor progression that are more likely to occur in dividing cells. Conversely, growth of ovarian surface epithelial cells ex vivo, freed from the constraints on proliferation in situ, might lead to malignant transformation. In this issue of the Journal (/), Godwin and colleagues present convincing evidence that repeated passage of rat ovarian surface epithelial cells in culture results in a loss of contact inhibition, as well as in the acquisition of anchorage-independent growth and the ability to form tumors in vivo after injection into athymic mice. Cytogenetic abnormalities have been observed in some cell lines both before and after transformation. Although the correlation between the chromosomal abnormalities described by Godwin et al. and the acquisition of a malignant phenotype is not precise, the potential for this type of analysis is clear. Spontaneous transformation in culture is thought to occur less frequently in cells taken from rats than in those taken from mice (/). Transformation in the absence of apparent carcinogens is not limited, however, to the ovarian epithelium, in that spontaneous transformation has also been observed with repeated passage of rat hepatocytes, fibroblasts, and tracheal epithelial cells (2-4). Whether spontaneous transformation in culture occurs with human ovarian surface epithelial cells remains to be determined. Investigators (5) have defined conditions that facilitate growth of apparently normal human ovarian epithelial cells and that should permit definitive studies with human tissues. Recent reports have begun to compare growth regulation in normal and malignant human ovarian epithelial cells. Preliminary data suggest that a majority of ovarian cancers are derived from single clones of cells. Consistent with studies in other malignancies, multiple genetic changes are likely to be required for malignant transformation of a normal epithelial clone. Different combinations of these changes might produce the same transformed phenotype. Certain changes might be observed more or less frequently in ovarian cancer than in tumors that

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arise from other sites. Aberrant regulation by peptide growth factors, activation of proto-oncogenes, or loss of tumor suppressor genes may all contribute to malignant transformation of ovarian epithelium. Epidermal growth factor (EGF) and its structural homologuetransforming growth factor alpha (TGF-a) bind to the EGF receptor, stimulating proliferation of normal epithelial cells from a number of sites, whereas transforming growth factor beta (TGF-P) binds to distinct receptors, inhibiting proliferation. Despite a low proliferative index, normal ovarian epithelial cells express both the EGF receptor and its ligand TGF-a. When normal human ovarian epithelial cells are grown in culture, signal transduction is maintained and cells respond regularly to exogenous EGF in anchorage-dependent assays of proliferation (6). By contrast, human ovarian carcinoma cell lines are quantitatively less responsive to EGF in anchorage-dependent assays, although most continue to express high-affinity EGF receptor (7). Addition of antibodies that neutralize TGF-a can inhibit growth of tumor cells that express TGF-a and EGF receptor, consistent with autocrine growth stimulation (8). Continued expression of EGF receptor is associated with a poor prognosis in patients with advanced ovarian cancer (9), possibly related to the persistence of an autocrine stimulatory loop. Godwin et al. (7) observed that rat ovarian epithelial cells which have gained the ability to grow in anchorage-independent assays show increased responsiveness to EGF. This observation requires confirmation with normal human ovarian epithelial cells after passage in culture or with ovarian cancer cells taken directly from patients. TGF-P may be an important factor constraining the growth of normal ovarian epithelium. The addition of TGF-p to cultures of normal human epithelial cells regularly inhibited anchoragedependent growth (10). Both TGF-p 1 and TGF-p 2 are expressed by normal human ovarian epithelial cells (10). TGF-P is also expressed by human granulosa cells (//). Consequently, TGF-P could participate in paracrine as well as autocrine growth inhibition. Autocrine growth inhibition by TGF-P appears to be lost in many ovarian cancer cell lines, where some fail to express the factor, others cannot activate it, and a majority have become refractory to inhibition by exogenous TGF-p (10). In one cell line that retained the ability to secrete active TGF-p and to respond to exogenous TGF-p, addition of anti-TGF-P antibodies stimulated anchorage-dependent growth (10). Thus, loss of autocrine growth inhibition by TGF-P might be an early step in the development of some, but not all, ovarian cancers. Godwin et al. (/) found that normal rat epithelium was not capable of anchorage-independent growth, and, consequently, any inhibitory activity of TGF-P could not be evaluated. After anchorage-independent growth was acquired in late passages, TGF-P generally inhibited the formation of tumor colonies in soft agar, but it did not consistently offset stimulation by TGFa. Studies of TGF-a and TGF-P on anchorage-dependent

Received March 16. 1992: accepted March 18. 1992. Duke University Medical Center, Durham. N.C. *Correspondence ro: Robert C. Bast, Jr., M.D., Box 3843, Duke University Medical Center, Durham, NC 27710.

Journal of the National Cancer Institute

growth of rat or human cells during early passages would be of particular interest. Loss of growth regulation by TGF-p might facilitate anchorage-dependent proliferation prior to the acquisition of anchorage independence. Alternatively, malignant transformation might relate to a dissociation of the stimulatory properties of TGF-cc from the inhibitory properties of TGF-p\ Other growth factors may be important, in that a growthstimulatory protein has been found in ovarian cancer ascites fluid that appears distinct from TGF-a or TGF-fi (72). Activation or overexpression of several proto-oncogenes has been detected in ovarian cancers. Despite an early report of somatic mutation in Ki-ras associated with an ovarian cancer (75), mutation or amplification of genes in the ras family has been observed in only 2%-12% of ovarian cancers evaluated in subsequent studies (14), a markedly lower frequency than that observed in pancreatic, colorectal, or lung cancers. Amplification of myc has been reported in up to 23% of ovarian cancers, but this does not appear to impact on prognosis (14). Of potentially greater significance, fms is expressed in approximately 50% and c-erbB-2 (HER-2/neu) in 86% of ovarian cancers (75). The fms proto-oncogene encodes the receptor for macrophage colony-stimulating factor (M-CSF). Normal ovarian epithelium and approximately 70% of ovarian cancers secrete biologically active M-CSF (16-18). Little, if any, fms gene product can be detected in normal ovarian epithelium, and its appearance in 50% of ovarian cancers could establish autocrine growth stimulation. Concomitant expression of M-CSF and fms is associated with a poor prognosis in ovarian cancer, correlating with advanced stage and high grade (16). Production of M-CSF by ovarian cancers could also lead to paracrine growth stimulation. M-CSF is a potent chemoattractant for macrophages. Macrophage products have long been known to stimulate growth of clonogenic ovarian tumor cells ex vivo. Recent studies have demonstrated that the macrophage-derived cytokines interleukin-1, interleukin-6, and TNF can all stimulate rather than inhibit the growth of ovarian cancer cells (79). Normal epithelium and approximately 70% of ovarian cancers express low levels of the HER-2/neu gene product pi85. Approximately 30% of ovarian cancers overexpress pi85, and this overexpression is associated with a very poor prognosis (20,21). Thus, each of the three novel molecular markers for a poor clinical outcome—EGF receptor, fms, and HER-2/neu—is a receptor transmembrane tyrosine kinase. Whether a poor prognosis relates to more aggressive tumor growth or to resistance to platinum-based chemotherapy remains to be resolved. The extracellular domains of these kinases can, however, serve as targets for monoclonal antibodies and immunotoxins (22). Pharmacologic manipulation of tyrosine kinase and tyrosine phosphatase activities may provide other attractive targets for therapy. Among the tumor suppressor genes, mutation and consequent overexpression of p53 have been found in approximately 50% of ovarian cancers (23). In contrast to breast cancer, p53 mutation does not appear to be associated with a worse prognosis. Mutations have, however, been found more frequently in advanced ovarian cancers than in early-stage disease. When mutant p53 genes have been sequenced, G->T transversions or transitions at CpG dinucleotides have not been particularly

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prevalent, which sets ovarian cancer apart from lung cancer and hepatoma on the one hand and colon cancer on the other. Loss of function of a tumor suppressor gene is thought to require loss or inactivation of both alleles or their products. One allele may be inactivated by somatic or germline mutation. The remaining wild-type allele can be inactivated by one of several mechanisms, including replacement by a reduplicated copy of the original mutation through chromosomal nondisjunction, mitotic recombination, or gene conversion (24). Potential suppressor genes have been sought at sites of chromosomal deletion or loss of heterozygosity using DNA markers for adjacent loci. The majority of epithelial ovarian cancers have an aneuploid DNA content (25). Of those cases with a diploid content, many have chromosomal deletions, duplications, and translocations rather than a normal 46,XX karyotype. Karyotypic analysis of ovarian cancer cells has been performed on specimens derived from cell lines, directly from ascites, and from solid ovarian tumor specimens after short-term tissue culture. These investigations have revealed a complex array of karyotypic changes but no consistent alteration common to all epithelial ovarian carcinomas (26). The complexity of karyotypic analysis in ovarian cancer may relate in part to the advanced stage or recurrent nature of the samples analyzed or to artifacts of cell culture systems. Even in a study of primary, untreated tumors, however, a mean of seven different abnormal chromosome patterns per tumor was observed (27). Abnormalities of chromosome 1 have been observed in a wide spectrum of solid tumors and are the most common karyotypic abnormalities associated with ovarian malignancy. The reported frequency of chromosome 1 rearrangements in ovarian cancer (>80%) is too high to be a random event due to chromosome size alone. Other karyotypic abnormalities frequently observed in ovarian cancer involve chromosomes 3, 6, and 11. Abnormalities of all chromosomes have been reported, but chromosomes 5 and 21 are the least frequently involved. A specific t(6; 14)(q21 ;24) translocation has been reported in a series of papillary serous adenocarcinomas (28) but has not been confirmed by subsequent studies. Interestingly, trisomy 12 has been observed as a sole abnormality in a variety of benign tumors, including fibromas and germ cell, granulosa cell, and epithelial tumors of the ovary (29). The significance of these observations in relation to ovarian malignancy is unclear, although duplications of 12p and 12q have been described in invasive epithelial ovarian cancer. The development of polymorphic genetic markers has recently provided an additional strategy of screening for genetic alterations by identifying loss of heterozygosity at specific allelic loci. Initial studies, directed by karyotypic findings, demonstrated losses involving chromosomal segments 3p, 6q, and 1 lp at nonrandom frequencies (30). Subsequently, allelotyping with a set of polymorphic markers to at least a part of each chromosome revealed a greater than 30% loss for loci on chromosomes 4p, 6p, 7p, 8q, 12p, 12q, 16p, 16q, 17p, 17q, and 19p but no loss for loci on chromosome 2p, 5q, or 21 (31). The pattern of loss of heterozygosity appears to correlate with tumor grade and histological type. Serous cystadenocarcinomas have a significantly greater frequency of loss of loci on 6q, 13q, and 19q than do nonserous tumors (31), whereas losses on chromosomes 3 and 11 are associated with high-grade tumors (32). A high incidence

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of loss of heterozygosity on chromosome 17 in ovarian cancer has been reported (33,34) and is of particular interest in view of the location of the p53 gene on 17p and the recent report of linkage of familial breast and ovarian cancers to the CMM86 locus on 17q21 (35). Our own data (36) are consistent with the existence of at least two deletion units on chromosome 17—one associated with the p53 gene and a second on the distal portion of 17q which is distinct from the linkage region reported in familial cases. Godwin et al. (7) observed loss of rat chromosome 5 in two transformed cell lines. It is therefore interesting to note that rearrangements of the homologous human chromosome 9p with loss of the distal region of 9p have been described in cytogenetic studies of human ovarian adenocarcinomas (37). Studies of this region in epithelial ovarian cancer for loss of heterozygosity have not yet been reported. The chromosomal abnormalities and loss of heterozygosity observed in human ovarian cancers might relate to genetic changes that develop spontaneously during cell proliferation or as a result of exposure to environmental carcinogens. If human ovarian epithelial cells undergo spontaneous transformation on repeated passage in culture, it will be of particular interest to determine whether at least some of the genetic changes observed in clinical material occur ex vivo.

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ovarian cancer. Cancer Genet Cytogenet 11:91-106, 1984 human epithelial ovarian malignancies. Gynecol Oncol 38:473—477. 1990 changes in ovarian cancer involving chromosomes 6 and 14. Cancer Res 40:4512-4518,1980

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factor-a acts as an autocrine growth factor in ovarian carcinoma cell lines. Cancer Res 52:341-347. 1992

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(//)

locus on chromosome 17ql2-q23. Lancet 338:82-83. 1991 (36) JACOBS IJ, WISEMAN R, O'BRIANT K, ET AL: LOSS of heterozygosity on

Journal of the National Cancer Institute

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ONLY YOU CAN PREVENT FOREST FIRES.

A PUBLIC SERVICE O f THE USDA FOREST SERVICE AND YOUR STATt FORESTERS

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