Incessant ovulation, inflammation and epithelial ovarian carcinogenesis: Revisiting old hypotheses

Molecular and Cellular Endocrinology 247 (2006) 4–21

At the Cutting Edge

Incessant ovulation, inflammation and epithelial ovarian carcinogenesis: Revisiting old hypotheses Jean S. Fleming a,∗ , Clare R. Beaugi´e b , Izhak Haviv c , Georgia Chenevix-Trench d , Olivia L. Tan e a

Eskitis Institute for Cell & Molecular Therapies, School of Biomolecular and Biomedical Sciences, Griffith University Nathan Campus, Nathan, Qld 4111, Australia b Department of Anatomy & Structural Biology, Otago School of Medical Sciences, P.O. Box 913, Dunedin, New Zealand c Peter MacCallum Cancer Centre, Ian Potter Centre for Cancer Genomics & Predictive Medicine, St. Andrews Place, East Melbourne, Vic. 3002, Australia d Cancer Genetics Laboratory, Queensland Institute of Medical Research, 300 Herston Road, Herston Brisbane, Qld 4029, Australia e Centre for Molecular Biotechnology, School of Life Sciences, Queensland University of Technology, Brisbane, Qld 4001, Australia Received 8 September 2005; received in revised form 30 September 2005; accepted 11 October 2005

Abstract Epithelial ovarian cancer (EOC) is often a lethal disease because in many cases early symptoms go undetected. Although research proceeds apace, as yet there are few reliable and specific biomarkers for the early stages of the disease. EOC is an umbrella label for a highly heterogeneous collection of cancers, which includes tumours of low malignant potential, serous cystadenomas, mucinous and clear cell carcinomas, all of which are likely to arise from a number of epithelial cell types and a variety of progenitor lesions. Many, but not all types of EOC are thought to arise from the cells lining ovarian inclusion cysts. In this review, we discuss the hypotheses that have driven our ideas on epithelial ovarian carcinogenesis and examine the morphological and genetic evidence for pathways to EOC. The emergence of laser-capture microdissection and expression profiling by microarray technologies offers the promise of defining these pathways more accurately, as well as providing us with the tools for earlier diagnosis. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Ovulation; Epithelial ovarian cancer; Inflammation; Ovarian inclusion cyst

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

∗

Epithelial cell types in the ovary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Histopathological evidence for the aetiology of EOC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic evidence for the aetiology of EOC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Incessant ovulation hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gonadotrophin hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hormonal hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The inflammation hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Expression profiling of EOC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biomarkers for EOC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Corresponding author. Tel.: +61 7 3735 3782; fax: +61 7 3735 37656. E-mail address: [email protected] (J.S. Fleming).

0303-7207/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2005.09.014

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Over 90% of ovarian cancers are described as epithelial in origin. Epithelial ovarian cancer (EOC) is a highly heterogeneous group of cancers, which can be classified into four main subtypes; serous, mucinous, endometrioid and clear cell, based largely on histological evidence (Korner et al., 2005). EOC is the fourth most common cause of death from cancer among women in the United States (Jemal et al., 2005) and has the highest mortality rate of the gynaecologic cancers (Alvarez et al., 1999; Ho, 2003). The 5-year survival rate is less than 40% (Persson, 2000), because of the presentation of the majority of cases at an advanced stage, but the aetiology and precursor lesions of even the major subtypes are poorly understood. While genetic studies are beginning to unravel different pathogenic pathways to the different EOC subtypes, our studies on the origin of ovarian inclusion cysts in mouse ovaries suggest there may be more than one epithelial cell of origin for serous ovarian adenomas (Tan et al., 2005). This review revisits the major hypotheses on the aetiology of EOC, in the light of new evidence from mouse models, mutation analysis and gene expression profiling.

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1. Epithelial cell types in the ovary The ovary is divided into an outer cortex and an inner medulla, which contains the major blood and lymphatic vessels. The point of entry into the ovary for these vessels is known as the ovarian hilus (Clement, 1987). Surrounding the ovary and continuous, at the hilus, with the peritoneal mesothelium, is a single layer of phenotypically uncommitted mesothelial cells known as the ovarian surface epithelium (OSE; Fig. 1) (Auersperg et al., 2001). The OSE, the mesothelial lining of the peritoneal cavity and the reproductive tract tissues, are all derived during development from the coelomic layer. The OSE develops from a region of epithelium overlying the gonadal ridge, whereas the m¨ullerian ducts, from which the reproductive tract tissues originate, arise from an invagination of the coelomic epithelium beside the gonadal ridge (Naora, 2005). It is therefore possible the OSE could show characteristics of m¨ullerian ductderived tissues, such as the fallopian tube, endometrium and cervix, on metaplastic transformation (Naora, 2005; Motta and

Fig. 1. Epithelial cells of the normal mouse ovary. All sections were stained with haematoxylin and eosin (H&E) or H alone. (A) The single cell mesothelial layer of the ovarian surface epithelium, which can be cuboidal or flattened, forms invaginations (inv) in older ovaries, often associated with corpora lutea (CL) formation. Ovary from a 9-month group-housed mouse; original magnification 400×. (B) The extraovarian tubules of the rete ovarii (arrows) from a 9-month breeder mouse are lined with cuboidal or columnar epithelium, which can be ciliated or contain clear cytoplasm; original magnification 40×. (C) Deep invagination in a 9-month mouse ovary, showing stratification of the epithelium lining an early inclusion cyst; original magnification 200×. (D) Connecting and intraovarian rete ovarii tubules (RO) are found at the hilus of the ovary and can be distinguished from lymphatics and blood vessels by their cuboidal or columnar cell structure; original magnification 400×.

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Van Blerkom, 1980; Risch, 1998). Epithelial ovarian cancers, specifically serous, endometrioid and mucinous, show histological and secretory properties of the epithelial and stromal tissues of the fallopian tube, endometrium and cervix, respectively, supporting this idea (Scully, 1977; Dubeau, 1999). The OSE cells have the potential to become epithelial or mesenchymal cells in response to ovulation or hormonal stimuli (Feeley and Wells, 2001). When they become cancerous, the cells appear to differentiate to a committed m¨ullerian phenotype, losing their plasticity and responsiveness to external cues (Feeley and Wells, 2001). EOC is different from other cancers in that the epithelium becomes more differentiated as it transforms, rather than less (Auersperg et al., 1997). For many decades, the OSE was described as the germinal epithelium and its cells were thought to be capable of differentiating to form new primordial follicles, complete with oocytes (Allen, 1923). As more became known about fetal development of the ovary, this idea was discarded and the post-natal ovary was regarded as incapable of producing new germ cells (Peters and McNatty, 1980). The recent publication of research pointing to the existence of ovarian stem or progenitor cells, capable of re-seeding the ovary with new oocytes (Johnson et al., 2004, 2005; Bukovsky et al., 2004), has turned reproductive biological dogma on its head. Given the increasing evidence that a variety of cancers, including breast cancer, may result from transformation of normal stem and progenitor cells (Woodward et al., 2005; Liu et al., 2005; Clarke et al., 2005; Li et al., 2003; Anderson and Clarke, 1999; Lochter, 1998), the existence of multi-potent stem cells capable of differentiating to an epithelial phenotype, should not be disregarded. However, until more is known about these proposed ovarian germ-line progenitor cells, including their extraovarian or ovarian origin, their differentiation pathways, their relationship to OSE cells and their response to factors thought to increase susceptibility to carcinogenesis, they must remain offstage in this review, awaiting their time in the limelight. Another source of ovarian epithelial cells is the reticulum of tubules known as rete ovarii (Fig. 1). The rete ovarii is a normal structure of the adult mammalian ovary and consists of a network of epithelial cell-lined tubules, which are thought to develop from the mesonephric tubules. During ovarian development, mesonephric cells migrate towards and into the undifferentiated gonad, resulting in the extension of the mesonephric tubules through the mesovarian (Czernobilsky et al., 1985; Byskov, 1978; Wenzel and Odend’hal, 1985). The function of the rete ovarii in the adult ovary is not well characterised and the tubules are generally assumed to be a functionless vestige of development. Byskov suggests the rete ovarii have an active role in initiating meiosis in the developing germ cells and proposes the rete ovarii is the origin of the granulosa cells (Byskov and Lintern-Moore, 1973; Byskov et al., 1977). The rete ovarii tubules exist in all species studied as three morphologically distinct groups; the intraovarian rete, the extraovarian rete and the connecting rete (Wenzel and Odend’hal, 1985). In humans, the major tubule of the extraovarian rete is also known as the epoophoron, which is located in the mesosalpinx. The intraovarian rete tubules are found near the hilus, in

the medulla of the ovary. Intraovarian rete are lined by cuboidal epithelia, whereas the connecting and extraovarian rete are lined by ciliated columnar epithelial cells (Wenzel and Odend’hal, 1985). The three tubule types are almost always connected. Morphology of the rete ovarii appears to change with pregnancy, being described as more luxuriant in pregnant women (Wenzel and Odend’hal, 1985). Changes in the rete ovarii throughout the oestrous cycle and pregnancy have also been reported in the beagle and cow (Wenzel and Odend’hal, 1985). These results suggest the rete ovarii is under endocrine influence. A secretory role has been hypothesised for the rete ovarii, as secreted materials have been found in the tubule lumen of cows (Archbald et al., 1971) and cats (Gelberg et al., 1984). It has been suggested the increased size of the rete ovarii during pregnancy in some species is caused by increased secretion from the cells lining the tubules. Morphological studies of ovaries from dogs, cows (Odend’hal et al., 1986) and sheep (Cassali et al., 2000) have revealed a physical connection between the rete ovarii and the infundibulum of the uterine tube, although the functional significance of this connection is unknown. Research on the structure and function of the rete ovarii has primarily focused on these structures in animals and consequently little is known about the human rete ovarii. Much of the limited information available on the human rete ovarii has come about because of the tendency for rete ovarii to be mistaken for the relatively common condition of endometriosis (Khan et al., 1999; Woolnough et al., 2000). Dilation or cysts of the rete ovarii have been described in almost all mammals studied, with the incidence of these cysts increasing with age (Clow et al., 2002; Thung et al., 1956; Altinoz and Korkmaz, 2004). These cysts are characterised by their location, which is always at the hilus or outside the ovary in the mesovarian region, associated with the ovarian ligament or adipose tissue. As mice age, the rete ovarii tubules dilate forming cystic structures (Fig. 2), with epithelial hyperplasia and increased levels of proliferation (Clow et al., 2002; Thung et al., 1956; Beaugi´e, 2005). Large cystic rete ovarii may compress adjacent structures, but do not appear to affect normal ovarian function (Clow et al., 2002; Sass and Rehm, 1994). These cysts may form after blockage of the tubules, preventing drainage of secreted fluids into the uterine tube (Odend’hal et al., 1986; Cassali et al., 2000). Cystic rete ovarii occur frequently in the ovaries of older women (Peters and McNatty, 1980; Rutgers and Scully, 1988; Nogales, 1995). The morphology of these structures is similar to that seen in other mammals, with cuboidal and columnar cells and clear cyst fluid (Rutgers and Scully, 1988). They are usually always diagnosed based on a hilar location and are often microscopic in size and benign (Nogales, 1995). In rare cases, these cysts can give rise to adenomas and adenocarcinoma of the rete ovarii (Rutgers and Scully, 1988; Nogales et al., 1997; Lee et al., 2001; Heatley, 2000). 2. Histopathological evidence for the aetiology of EOC EOC appears to arise via one of at least two pathways; spontaneously and aggressively, with no precursor lesion (Type 2), or by slower development from an inclusion cyst to a benign

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Fig. 2. Haematoxylin-stained inclusion cysts in ovaries of 9-month breeder or incessantly ovulated mice, containing papillae (A) and/or secretory cells (B). (C) Aspirated serous inclusion cyst lined in places with cells resembling signet ring or hobnail cells (arrows). (D) Single layer of secretory cells lining a cortical cyst. Cilia are frequently present on cyst surface cells (*). Original magnifications: (A) 200×; (B) 1000×; (C) 200×; (D) 1000×.

adenoma or cystadenoma of low malignant potential (LMP), through to metastatic adenocarcinoma (Type 1). Benign inclusion cysts may result from invaginations of OSE into the ovarian stroma (Feeley and Wells, 2001) and/or from OSE becoming entrapped within the stroma following ovulation (Radisavljevic, 1977). However, the presence of inclusion cysts appears not to be related to ovulation frequency, as polycystic ovaries have a high frequency of inclusion cysts (Resta et al., 1993), suggesting that they form from invaginations independently from ovulation. Studies of ovaries removed prophylactically from women with a family history of ovarian cancer and contralateral ovaries from women with EOC also show an increase in the number of inclusion cysts, cellular abnormalities and metaplasia in the epithelia lining the cysts (Resta et al., 1993; Okamura and Katabuchi, 2001; Salazar et al., 1996). However, other research has found no such differences (Werness et al., 1999). Inclusion cyst frequency has also been found to be positively associated with age in women with unilateral ovarian cancer (Mittal et al., 1993). Nuclear and cytologic changes, such as loss of polarity, unusual nuclear structures and epithelial stratification have been observed in OSE adjacent to ovarian carcinomas and in OSE of women with a family history of EOC (Salazar et al.,

1996; Werness et al., 1999; Deligdisch and Gil, 1989; Plaxe et al., 1990). Another study found atypical cells only in inclusion cysts adjacent to ovarian cancer or in the contralateral ovary, whereas normal cells were seen in inclusion cysts adjacent to borderline tumours or in control ovaries (Hutson et al., 1995). The phenotypic change to m¨ullerian-like epithelium followed by metaplasia, observed in many cysts, results in a serous phenotype and it is possible that undefined molecular events occurring in such a cyst may initiate neoplastic change. Benign serous cysts have a higher likelihood of becoming malignant than mucinous cysts (Mulligan, 1976; Singer et al., 2002). Studies on microscopic carcinomas and using transvaginal ultrasonography have shown a subset of ovarian cancers can also arise de novo, without any immediate cystic precursors (Bell and Scully, 1994; Horiuchi et al., 2003), either directly from the OSE or from the rete ovarii (Dubeau, 1999; Mckay, 1962). The speculation that EOC can arise from the peritoneal epithelium or secondary m¨ullerian system, rather than the OSE (Dubeau, 1999), would also explain the presence of ovarianlike cysts seen in the peritoneum of women who have had their ovaries removed (Sightler et al., 1991) or who have normal ovaries (Foyle et al., 1981). Evidence against this origin for EOC includes the observation that when cancer is detected early, it is

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located in the cortex near the OSE, not in the medulla or near the hilus of the ovary (Feeley and Wells, 2001). Although Dubeau (1999) argues that accurate evaluation of the anatomical position of these cysts is difficult, because of their size, the distortion of the ovary and lack of serial sectioning, Feeley and Wells (2001) conclude the majority of EOCs originate either from the OSE or from inclusion cysts. Our observation that incessant ovulation leads to an increase in non-hilar, cortical inclusion cysts in the mouse ovary, while suggesting the proportion of cysts from each location may vary with species, does support this conclusion (Tan et al., 2005). 3. Genetic evidence for the aetiology of EOC Genetic analyses can help to unravel the origins and heterogeneity of EOC at many levels. It is well recognised that family history of ovarian or breast cancer is one of the major risk factors for EOC (Easton et al., 1996), and some of the highly penetrant genes that contribute to this genetic susceptibility have been identified. Germ-line mutations in BRCA1 and BRCA2 confer an average cumulated risk of EOC by age 70 of 39% and 11%, respectively (Antoniou et al., 2003). Invasive serous EOC, and to a lesser extent, endometrioid invasive EOC, occur at increased frequency in BRCA1 and BRCA2 mutation carriers, but not in LMPs or other histologic subtypes of EOC (Risch et al., 2001). Mutations in the hereditary non-polyposis colorectal cancer genes, MSH2 and MLH1, also increase the risk of invasive EOC substantially, particularly of the endometrioid subtype (Watson et al., 1990). As well as the highly penetrant mutations in these susceptibility genes, it is likely that other genes exist with common, or perhaps rare, polymorphic alleles that have more subtle effects on risk of EOC. Many association studies have been published evaluating the role of common single nucleotide polymorphisms (SNPs) and risk of EOC, and many of these studies suggest some heterogeneity of risk for different subtypes of EOC (Berchuck et al., 2004; Spurdle et al., 2001a,b). However, association studies of this type are plagued by publication of many false positive associations that cannot be independently replicated (Wacholder et al., 2004). One exception to this might be the association between a SNP in the promoter of the progesterone receptor and risk of the endometrioid and clear cell subtypes of EOC, which has been replicated in two independent studies since the initial publication (Berchuck et al., 2004). In addition to the evidence that germ-line variants confer differential risks for different subtypes of EOC, there are many reports that the genetic and epigenetic changes that occur during ovarian tumourigenesis differ among the different subtypes of EOC. Furthermore, these somatic changes in sporadic tumours can help to unravel the relationships between different subtypes of EOC. There are several genes, such as CDKN2A (Shih et al., 1997), PTEN (Obata et al., 1998), TGFβRI (Chen et al., 2001), TGFβRII (Lynch et al., 1998), km23 (Ding et al., 2005), CTNBB1 (Palacios and Gamallo, 1998; Wright et al., 1999) and PIK3CA (Campbell et al., 2004), which are occasionally mutated in EOC and for many of these genes the mutations occur most often in endometrioid or clear cell subtypes. By contrast, the tumour sup-

pressor gene, TP53, is mutated in about 50% of invasive serous EOC, but only rarely in other histological subtypes, or in LMP tumours (Milner et al., 1997; Teneriello et al., 1993). Mutations of the KRAS oncogene are found in about 30% of LMP serous tumours, but they are rarely observed in invasive serous tumours, which is strong evidence that some or all invasive serous EOC do not derive from their LMP counterparts (Milner et al., 1997; Teneriello et al., 1993). This would be consistent with the finding that BRCA1 mutation carriers do not appear to have an increased risk of serous LMP EOC, but only serous invasive EOC (Bjorge et al., 2004). In contrast, identical BRAF and KRAS mutations have sometimes been found in benign epithelium and the adjacent serous and mucinous ovarian LMP tumours (Ho et al., 2004; Mandai et al., 1998), which suggests that BRAF and KRAS mutations are very early events in tumour development, and that serous and mucinous LMP tumours can arise from benign epithelia. As well as occurring in about 50% mucinous LMPs, KRAS mutations have also been frequently found in invasive mucinous EOC (Enomoto et al., 1991; Mok et al., 1993), which suggests that, unlike serous EOC, invasive mucinous tumours may derive from their LMP counterparts (Mandai et al., 1998). At another level, loss of heterozygosity in LMP tumours is at a similar level to that in malignant mucinous tumours (Watson et al., 1998) and transitions between tumours of LMP and malignancy are found histologically in 80% of cases. Furthermore, mucinous tumours with benign components are diagnosed 7–16 years on average earlier than those without benign areas, implying a temporal relationship (Feeley and Wells, 2001). However, the similar rate of chromosomal lesions in benign and LMP conditions, suggests those mutations themselves could, sooner or later, lead to cancer. Loss of heterozygosity analyses also support the concept that at least some LMP tumours may originate in benign cystadenomas, as the same targets of allelic loss have been reported in both types of EOC (Chenevix-Trench et al., 1997; Thomas et al., 2003). There is also evidence from loss of heterozygosity analyses, as well as numerous histological observations, that endometrioid and clear cell ovarian carcinomas can sometimes arise through malignant transformation of endometrioid lesions (Jiang et al., 1998). In summary, there are no detailed and validated models for the development and progression of ovarian tumours, the relationship between the various forms, or the sequence of genetic changes involved. However, there is clinicopathological and molecular evidence for a model in which epithelial tumours are broadly divided into two types, Type 1 and Type 2 (Donninger et al., 2004; Russell and McCluggage, 2004). Type 1 tumours are low-grade and develop through a hyperplastic process from the OSE to a benign lesion (such as an inclusion cyst), and then through a LMP tumour and into the invasive form. In contrast, Type 2 tumours are high-grade tumours, predominantly high grade serous carcinoma, for which precursor lesions have not been identified, and so they are thought to develop directly from the OSE. Different mutations and chromosomal abnormalities have been associated with the two pathways (Korner et al., 2005; Bell, 2005).

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In order to understand the underlying causes and types of EOC, it is useful to return to the major hypotheses that have attempted to relate the epidemiology to reproductive physiology. These include the incessant ovulation hypothesis (Fathalla, 1971), the gonadotrophin hypothesis (Cramer and Welch, 1983) and the hormonal hypothesis (Risch, 1998). The evidence for and against each of these theories is summarised below. 4. Incessant ovulation hypothesis The incessant ovulation hypothesis proposes that ovulation traumatises the ovarian surface, because rupture of the ovulating follicle damages the OSE, requiring immediate repair. Over time, this process of continuous damage and OSE proliferation to repair the wound, places strain on the OSE, increasing the chance of errors occurring during replication (Fathalla, 1971). Therefore, women with high lifetime ovulation numbers are at an increased risk of developing EOC. Over the last century incidence of EOC has increased, as have the average number of ovulations experienced in a lifetime (Banks et al., 1993). A century ago women often had many children, and thus ovulation was frequently suppressed either by pregnancy or lactation. It has been estimated that women living in the 19th century ovulated 40–50 oocytes, whereas modern women are thought to ovulate 400–500 oocytes in their reproductive lifetime (Banks et al., 1993). Fathalla’s hypothesis is further supported by evidence of a decrease in EOC risk with decreased lifetime ovulation number, through pregnancy or oral contraceptive pill use (Shu et al., 1989; Whittemore et al., 1992; Whittemore, 1993; Rodriguez et al., 1998a; Tortolero-Luna et al., 1994; Adami et al., 1994; Risch et al., 1983, 1994; Riman et al., 2002; Nasca et al., 1984; Negri et al., 1991; Akhmedkhanov et al., 2001; Titus-Ernstoff et al., 2001; Modan et al., 2001; Tavani et al., 2000). In addition, the longer ovulation is suppressed (from more pregnancies or longer use of the oral contraceptive pill), the lower the risk of developing EOC. When the total lifetime ovulation number was calculated for women who had EOC and for those that did not, a significant correlation between high total lifetime ovulation number and the occurrence of cancer was found (Purdie et al., 2003). Similar studies investigating an association between ‘ovulatory age’ and cancer risk have also found this to be the case (Whittemore, 1993; Casagrande et al., 1979; Moorman et al., 2002; Beard et al., 2000). The incessant ovulation hypothesis is further supported by fact that epithelial ovarian tumours are rare in other non-primate mammals (Land, 1993; Gondos, 1975), possibly because most animals do not normally reach high total ovulation numbers. Humans are one of the few species that continuously cycle. Many large farm animals are seasonal breeders and will only ovulate at certain times of the year, whereas some smaller animals are reflex ovulators and will only ovulate if mated. Alternatively, some animals will only cycle in the presence of a male (Austin and Short, 1984). It is therefore thought that other species can never reach an ‘ovulatory age’ high enough to develop cancer. The only species other than humans to frequently develop EOC are hens, specifically those that have been hyperovulated to produce

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eggs (Fredrickson, 1987; Wilson, 1958), adding further support for the incessant ovulation hypothesis. However, there are some who feel this model is too simplistic (La Vecchia et al., 1983; Siskind et al., 2000). The protection gained from pregnancy or even short-term oral contraceptive use is far superior to that expected if ovulation number alone were considered (Siskind et al., 2000; Gwinn et al., 1990; Greer et al., 2005). Use of the oral contraceptive pill reduces the relative risk of developing EOC by 7% for each year of use, with protection persisting beyond 15 years of exposure (Siskind et al., 2000). Each month of pregnancy has shown to reduce EOC risk by 2.6% and each month of breast-feeding by 2.4% (Gwinn et al., 1990). In addition, use of the progestin-only contraceptive pill (which does not suppress ovulation) has been shown to be even more protective against EOC than the combined oral contraceptive pill (Risch, 1998). There is also evidence that women with some forms of anovulatory infertility, such as polycystic ovarian syndrome, are at increased risk of developing EOC (Schildkraut et al., 1996). Other forms of infertility also appear to increase the risk of developing EOC. It is well documented that nulligravid women are at increased risk of developing EOC (Risch et al., 1983, 1994; Riman et al., 2002; Purdie et al., 1995). However, it has been reported that women who have never been pregnant because of infertility have a 40% higher rate of developing EOC than women who have never attempted to become pregnant (Rodriguez et al., 1998b). 5. Gonadotrophin hypothesis The gonadotrophin hypothesis states that excessive gonadotrophin exposure increases oestrogenic stimulation of the OSE, possibly leading to malignant transformation (Cramer and Welch, 1983). Gonadotrophins could act either directly on the OSE, enhancing transformation, or indirectly by stimulating oestrogen production (Mohle et al., 1985). Gonadotrophin levels increase with increasing age and are particularly high during menopause, consistent with the age-specific rates of EOC (Beltsos and Odem, 1996). Both pregnancy and the oral contraceptive pill lower circulating pituitary gonadotrophin levels (Risch, 1998), although significant increases in levels of both oestrogen and human chorionic gonadotrophin (hCG) occur during pregnancy (Risch, 1998). Women with polycystic ovarian syndrome frequently have a high luteinising hormone to follicle stimulating hormone ratio, along with an elevated risk of developing EOC (Schildkraut et al., 1996), lending support for the gonadotrophin hypothesis at the expense of the incessant ovulation hypothesis. If the gonadotrophin theory holds true, a significant increase in ovarian cancer risk with gonadotrophin use for in vitro fertilisation or infertility would be expected, however the evidence here is equivocal. A significantly increased risk for tumour development has been reported in infertile women who were given gonadotrophin treatment, compared with infertile women who were not (Rossing and Daling, 1994). However, actual cases of EOC appear not to differ between these groups (Harris et al., 1992). Rats stimulated with gonadotrophins had a higher incidence of ovarian cyst formation compared with the control

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group, although none of the rats developed EOC (Celik et al., 2004). Some epidemiological data conflict with the gonadotrophin hypothesis. Lactating women have raised levels of circulating FSH, yet the epidemiology suggests breast-feeding is protective (Adami et al., 1994; Harris et al., 1992). Conversely, some EOC patients have been shown to have lower levels of gonadotrophins (Risch, 1998; Ness and Cottreau, 1999) and women with particularly low levels of circulating gonadotrophins are at an increased risk of developing EOC (Daly and Obrams, 1998). 6. Hormonal hypothesis The hormonal hypothesis attempts, in part, to resolve these conflicts and proposes that excess androgen stimulation of the OSE leads to increased risk of cancer, whereas progesterone stimulation of the OSE is protective of EOC (Risch, 1998). Androgens are produced in the ovary by the developing follicles at higher rates than estrogens (Risch, 1998) and are the principal sex steroids present within the fluid of growing follicles (McNatty et al., 1979). It has been proposed that epithelia-lined inclusion cysts located near developing follicles may be exposed to high levels of androgens (Risch, 1998). Androgen receptors have been detected in OSE and rete ovarii epithelial cells of canine, primate and human ovaries (Saunders et al., 2000; Vermeirsch et al., 2001; Edmondson et al., 2002) and studies in guinea pigs have shown androgens stimulate proliferation in both inclusion cysts and OSE (Silva et al., 1997; Bai et al., 2000). However, androgen stimulation of human OSE cultures has no proliferative effect (Karlan et al., 1995). One of the key characteristics of polycystic ovarian syndrome is elevated androgen levels and these may contribute to increased risk of EOC in this condition (Schildkraut et al., 1996). A link has also been observed between increased EOC risk and a history of acne and hirsutism, both symptoms of elevated androgen levels (Wynder et al., 1969). Finally, the oral contraceptive pill, which is well known as protective, suppresses ovarian testosterone production (Gaspard et al., 1983). Not only does the oral contraceptive pill decrease levels of androgens, but it also contains high levels of progestins, with potencies considerably higher than that of progesterone. Long-term exposure to high levels of progestins could be the explanation for the pill’s significant protective effect (Ho, 2003; Risch, 1998). In addition, the progestin-only pill, which does not suppress ovulation, decreases EOC risk to the same or greater level than that seen with the combined contraceptive pill (Rosenberg et al., 1994). During pregnancy progesterone levels are very high, dwarfing circulating androgen concentrations. It has been suggested that the protection gained from pregnancy is through the 8–9 months of continuously high progesterone levels, rather than through suppression of ovulation (Risch, 1998). An inhibitory effect of progesterone on cell proliferation has been demonstrated in cell cultures of OSE from some pre- and post-menopausal women (Ivarsson et al., 2001), although similar studies have found no effect of progesterone on proliferation, either in the rabbit (Bai et al., 2000) or human (Karlan et al., 1995). Administration of progesterone to sheep OSE cultures

resulted in a decrease in basal and oestradiol-17␤-stimulated proliferation rates, as well as up-regulation of p53 tumour suppresser gene expression (Murdoch, 2002). Progesterone has also been shown to induce apoptosis and up-regulate p53 expression in human EOC-derived cell lines (Bu et al., 1997), suggesting the steroid is capable of inhibiting cell division, even after transformation. When administered to monkeys, progesterone can induce apoptosis in the OSE in vivo (Rodriguez et al., 1998a). Hens treated with progesterone have a decreased incidence of EOC, supporting the hypothesis that progesterone induces apoptosis of damaged OSE cells (Fredrickson, 1987; Rodriguez et al., 2001). Reversible formation of ovarian inclusion cysts has been reported frequently in breast cancer patients receiving tamoxifen chemotherapy (Cohen et al., 2003). Tamoxifen is a non-steroidal anti-oestrogen in breast tissue, but appears to be oestrogenic in tissues such as ovary (Mourits et al., 1999; McCluggage and Weir, 2000). Treatment with tamoxifen can raise 17␤-oestradiol serum levels significantly and is associated with the development of bilateral ovarian cysts in 80% of treated patients (Cohen et al., 1999). Breast cancer patients receiving tamoxifen only develop ovarian cysts if their ovaries are able to respond to FSH stimulation as shown by oestradiol production (Mourits et al., 1999) and the cysts appear to resolve in many patients on cessation of treatment (Cohen et al., 2003; Inal et al., 2005). There is no evidence that tamoxifen exposure is associated with an increase in benign or malignant primary or metastatic ovarian neoplasm (McGonigle et al., 1999). 7. The inflammation hypothesis The ovulatory process resembles an inflammatory reaction, with an infiltration of leukocytes and production of inflammatory mediators such as cytokines, closely associated with extensive tissue remodelling (Bonello et al., 1996). As the pre-ovulatory follicle expands, the theca externa and OSE cells on the surface first proliferate to accommodate the growing follicle and then undergo apoptosis as the follicle wall thins (Murdoch, 1995; Murdoch et al., 1999). The connective tissue layers of the tunica albuginea and theca externa must be weakened before the follicle wall can break open. These changes are probably dependent on release of collagenases and proteases characteristic of tissue response to inflammatory reactions. Extracellular matrix (ECM) proteolysis is also an important step in the growth and metastatic spread of any tumour (Bonello et al., 1996). Initiation of follicle wall breakdown stimulated by the pre-ovulatory luteinising hormone surge induces progesterone receptor synthesis in granulosa cells (Clemens et al., 1998). The rupture of the ovulatory follicle and release of the oocyte results in an increase in progesterone synthesis by the granulosa cells of the ovulated ovary, now freed from control by oocyte paracrine factors (Hunter et al., 2005). Production of progesterone around ovulation helps maintain DNA integrity in OSE cells damaged in the wave of apoptosis and DNA fragmentation prior to ovulation (Murdoch, 1995, 1994). Inflammatory stimuli induce 11␤ hydroxysteroid dehydrogenase-1 expression and activity in granulosa cells

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(Tetsuka et al., 1999) and OSE cells (Yong et al., 2002), causing the reversible metabolism of cortisone to cortisol (Tannin et al., 1991; Stewart and Mason, 1995). Cortisol may act to counteract the inflammation arising at ovulation (Yong et al., 2002; Andersen and Hornnes, 1994; Escher et al., 1997; Hillier and Tetsuka, 1998; Rae et al., 2004). The main mediator of the inflammatory reaction may be nitric oxide released from invading leukocytes, which acts as a potent vasodilator and mediates interleukin-1 beta-directed tissue remodelling events within the ovary (Bonello et al., 1996). However, constitutively expressed endothelial nitric oxide synthase (e-NOS) and inducible i-NOS are also present in the stromal cells and theca of the developing follicles and are up-regulated by hormonal stimulation at ovulation (Zackrisson et al., 1996). Thus, each round of ovulation is associated with leukocyte invasion, release of nitric oxide and inflammatory cytokines, vasodilation, DNA repair and tissue remodelling. Differentiation of the ovulated follicle into the new corpus luteum also involves substantial angiogenesis (Zackrisson et al., 1996). The persistence of genetic damage caused by inflammatory factors may therefore be an important factor in transformation of damaged OSE cells to EOC (Ness et al., 2000). Hardiman et al. (2000) have suggested that genital tract infections may be one factor that can give rise to both infertility and ovarian cancer in women who are unable to conceive despite medical treatment. Infection, leading to an acute inflammatory response, may block the endogenous ovulatory inflammatory response and inhibit ovulation. Primary ovarian failure with lack of oestrogen production, may also lead to an increase in gonadotrophin release from the anterior pituitary and consequently a higher exposure of the ovary to both gonadotrophins and androgens (Cramer and Welch, 1983). The risk of EOC is reduced in women who are consistent users of low dose aspirin, acetaminophen or non-steroidal anti-inflammatory agents for at least 6 months (Altinoz and Korkmaz, 2004; Akhmedkhanov et al., 2001). These anti-inflammatory agents are thought to act via a shared pathway, dependent on the suppression of transcription factor NF-kappaB activity, which may subsequently decrease transcription of growth factors, chemokines and proteases, including cyclooxygenase (COX)-2, vascular endothelial growth factor (VEGF) and various interleukins and chemokines. These can induce angiogenesis, invasion, autocrine growth loops and resistance to apoptosis (Altinoz and Korkmaz, 2004; Akhmedkhanov et al., 2001). Chemokines such as monocyte chemoattractant peptide 1 are potent attractors of leukocytes, particularly macrophages and T cells and are released just prior to ovulation (Altinoz and Korkmaz, 2004). Ovarian tumours have also been reported to contain infiltrating macrophages and T cells (Merogi et al., 1997; Negus et al., 1997), probably attracted by the production and release of chemokines (Burke et al., 1996; Negus et al., 1995). Chemokines are capable of initiating an immune response against malignant cells (Parmiani, 1990; Rollins and Sunday, 1991; Peoples et al., 1993; Goedegebuure et al., 1997) and, indeed, the presence of T cells in ovarian cancer patients has been shown to be significantly associated with increased progression-free time and overall survival (Zhang et al., 2003). Consistently, a chemokine that recruits

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regulatory (immune attenuating) T cells (CD4/CD25/FOXP3 positive) is associated with worse outcome in ovarian cancer (Curiel et al., 2004). On the other hand, macrophages can stimulate tumour growth and/or vascularisation (Malik and Balkwill, 1991; Mantovani et al., 1992). COX-2 expression is stimulated in normal ovary by tumour necrosis factor-alpha, but in EOC this signalling is uncoupled and COX-2 expression is frequently lost (Yang et al., 2005). Loss of COX-2 also correlates closely with morphological transformation of the epithelium to a neoplastic phenotype. This suggests COX-2 expression does not contribute directly to EOC malignancy, unlike in other epithelial cancers (Yang et al., 2005). There have been numerous epidemiological studies linking exposure to inflammatory factors such as talc or asbestos particles, to higher EOC risk (Purdie et al., 1995; Ness and Cottreau, 1999; Daly and Obrams, 1998; Baker and Piver, 1994; Chen et al., 1992). Direct evidence for such a connection is somewhat harder to find. A study on ovaries from women reporting frequent use and no use of perineal powdering with talcum powder, revealed talc particles on the surface of all ovaries, definitively demonstrating that the particles can reach the upper genital tract and suggesting post-natal use of talc may have contributed to the finding (Heller et al., 1996). Two early studies in the rat showed inflammatory and metaplastic effects on the OSE from talc injected under the bursal capsule (Hamilton et al., 1984), as well as demonstrating that talc particles could migrate from the perineum to the ovarian surface (Henderson et al., 1986). We have also demonstrated the passage of talc particles to the ovarian surface of mice, after perineal dusting once a day for 2 months (N. Armanasco and J.S. Fleming, unpublished data; Fig. 3), but this study failed to show any significant morphological changes in the OSE in such a short time frame. Although environmental factors, such as talc, can clearly access the ovarian surface, the significance of the inflammatory response to their presence has yet to be demonstrated. 8. Expression profiling of EOC The four hypotheses on the aetiology of EOC may point towards causal factors, but the main obstacle to improving the mortality rate of this so often fatal disease is our lack of ability to identify the cancer early, to fight it more effectively and to prevent its appearance. Microarrays have proved themselves useful in classification of tumours into distinct, clinically relevant subtypes and the prediction of clinical outcomes. Some classification studies are now moving from the laboratories of basic investigators to large-scale clinical trials. As measuring expression of all genes in the human genome becomes a standard procedure, global gene expression analysis may become the primary tool by which we can achieve personalised treatments. This will be particularly important in the prediction of response to chemotherapy (Jazaeri et al., 2005). The advantage of microarrays is that you obtain a comprehensive parallel record of genes that are expressed together in a condition-dependent manner. This in turn allows the monitoring of groups of genes that respond in parallel to similar signal transduction pathways (Segal et al., 2004; Ihmels et al., 2002).

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Fig. 3. Scanning electron micrograph (SEM) of talc particles (arrow) on the ovary of a mouse subjected to a daily dusting of talc on the perineum for 12 weeks. Left inset: SEM of talcum powder used for dusting mice. Right inset: SEM of talc particles on the OSE of a different ovary, showing evidence of particle engulfment by epithelial cells (arrow).

The heterogeneity of EOC, compared with other types of cancer, makes interpretation of the results extremely difficult, since it is likely that multiple independent molecular mechanisms may underlie the same phenotype in different samples. For the most part, microarray results are more interpretable, beyond endless gene lists, when the experiments conducted have clearly defined aims and a strategy that allows a focus on the genes that relate to these aims. Here we will review recent examples of how genomic technology can be used to improve our understanding, detection and treatment of ovarian cancer, with emphasis on studies where the aims were clearly outlined and pursued. 9. Biomarkers for EOC The main cause of fatality in EOC is believed to be the late stage that patients present with symptoms. Therefore, the incentive for early detection is enormous. Microarrays are ideally positioned to provide data on potential biomarkers for this purpose and indeed, a number of such genes have been identified so far. Since the ultimate aim is a non-invasive test, focus on upregulated genes encoding transmembrane receptors and secreted proteins is crucial. The main technical obstacles in EOC detection are the ambiguity of the cellular origin of ovarian cancer and the small numbers of possible progenitor cells. The OSE amounts to no more than 300,000 cells in the adult body. One possibility would be to compare late and early stage ovarian tumours, with the rational that the test is only really needed to identify the deadly advanced cases. Surprisingly, many of the expression and genomic profiles obtained identify no differentially expressed genes between early and late stage tumours (Shridhar et al., 2001, 2002) and extremely small numbers of coincidentally expressed genes, between tumours of LMP and invasive tumours (Gilks et al., 2005; Meinhold-Heerlein

et al., 2005; Warrenfeltz et al., 2004). These expression profiles support the ideas expressed previously, that in some cases benign and LMP tumours are not precursors of invasive tumours (Chenevix-Trench et al., 1997; Kurman and Trimble, 1993). In order to compare tumour expression to that in OSE, one needs to obtain sufficient numbers of these cells. Since the tumour profile typically is generated from fresh frozen, total resected mass, generating a comparable sample from normal OSE is difficult and whatever adaptation is chosen for comparison will make a fundamental difference to the genes identified (Zorn et al., 2003). One solution is laser-capture microscopy (LCM)-mediated microdissection of OSE and EOC cell populations, combined with SAGE or microarray analysis to generate a list of candidates (Table 1), that are then validated by real-time PCR (Hough et al., 2000, 2001). Another solution is to correlate patterns of gene expression in the different histotypes of EOC, with the normal tissues each histotype resembles. A recent profiling study compared serous, mucinous, endometrioid and clear cell EOC with normal OSE brushings, as well as fallopian tube, endometrium and colon (Marquez et al., 2005). When compared with normal OSE, the changes in gene expression noted in serous EOC correlated with those found in fallopian tube, but not in other normal tissues. Similarly, differences between mucinous EOC and normal OSE correlated with those in normal colon, and differences between both endometrioid and clear cell cancers and OSE correlated with those in normal endometrium (Marquez et al., 2005). This type of study begins to identify the specific molecular alterations and pathways that lead to the different EOC histotypes, as well as identifying important histotype-specific biomarkers (Table 1). Alternatively, EOC-specific genes are sought within cell cultures, which can be obtained for both normal OSE and EOC cells (Euer et al., 2005). Because of the problem of access to the nor-

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Table 1 Genes identified as up-regulated in EOC, and therefore of potential use as biomarkers, in a range of studies using microarray or SAGE techniques Potential biomarkers

Tissues/cells compared

References

Apolipoprotein J/clusterin Apolipoprotein E Ceruloplasmin

Primary cancers, ovarian surface epithelial cells and cystadenoma cells Laser-capture cell microdissected EOC and SAGE analysis

Hough et al. (2000, 2001)

Cell adhesion molecule L1 CAM Notch ligand Jagged 2 Secreted polypeptide Neuromedin U

11 ovarian tumour cell lines and 2 immortalised OSE cell lines

Euer et al. (2005)

␤8 integrin subunit Bone morphogenetic protein-7 Cellular retinoic acid binding protein-1 Claudin-4 Collagen type IX ␣2 Forkhead box J1 S100 calcium-binding protein A1

20 ovarian carcinomas, 17 ovarian carcinomas metastatic to the omentum and 50 normal ovaries

Hibbs et al. (2004)

Prostasin

3 ovarian cancer cell lines and from 3 normal human OSE cell lines

Mok et al. (2001)

CD24 Claudin-3 Claudin-4 Kallikrein 6 (protease M) Kallikrein 10 Ladinin 1 Laminin Lipocalin 2 Matriptase (TADG-15) Osteopontin S100A2 SERPIN2 (PAI-2) Stratifin Tumour-associated calcium signal transducers 1 and 2 (TROP-1/Ep-CAM, TROP-2)

10 primary OSPC cell lines, 2 established OSPC cell lines (UCI-101, UCI-107) and 5 primary normal OSE epithelial cultures

Santin et al. (2004) and Shvartsman et al. (2003)

Kallikrein 6 Kallikrein 10

Epithelial ovarian carcinomas and normal ovarian tissue

Welsh et al. (2003)

Trefoil factor 1 (TFF1) Anterior gradient 2 (AGR2) Lectin, galactoside-binding, soluble, 4 (LGALS4) Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM6) Cathepsin E (CTSE)

Normal OSE brushings used as reference standard Mucinous EOC compared with normal colon Serous EOC compared with normal fallopian tube Endometrioid and clear cell EOC compared with normal endometrium

Marquez et al. (2005)

Claudin-3 Claudin-4 Epithelial cell adhesion molecule/GA733-2 Folate receptor 1 Glutathione peroxidase 3 (GPX3) HE4 IGF-binding protein 2 Kop protease inhibitor Matrix gla protein Mesothelin Mucin-1 S100A2 Secretory leucocyte protease inhibitor Signal transducer and activator of transcription 1 Tissue inhibitor of metalloproteinase 3

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mal controls, such studies depend on downstream immunohistochemical validation in primary material. L1CAM is expressed predominantly in ovarian carcinomas, in borderline tumours to a lesser extent and very rarely in ovarian non-epithelial types of cancer. Similarly, the potential markers found by Hibbs et al. (2004) are all over-expressed in EOC (Table 1). Statistical analyses showed that the ␤8 integrin subunit, claudin-4 and S100A1 provided the best distinction between ovarian carcinoma and normal ovary and served as the best candidate tumour markers among the seven genes identified in one study (Table 1) (Hibbs et al., 2004). Biomarkers may be present at higher levels in the cancer tissue as a result of increased signalling of a specific pathway in the tumour microenvironment (Donninger et al., 2004; Hough et al., 2001), or as a result of a specific chromosomal amplification that leads to selective advantage. In such a case, the primary validation may use a combination of expression profiling with comparative genome hybridization (CGH) (Shridhar et al., 2001; Israeli et al., 2005; Scorilas et al., 2003). Ostensibly, some typical EOC markers may mark amplified genome loci, not because those genes are selectively advantageous, but rather because a gene in their genomic vicinity is also amplified. For example, folate receptor, whose selective advantage to EOC has long been sought (Bottero et al., 1993), resides in the centre of an amplicon next to Cyclin D1 (Shridhar et al., 2001), whose advantage to growth is easier to explain (Hanahan and Weinberg, 2000). Other EOC putative markers include prostasin (Mok et al., 2001), kallikrein KLK10 (Santin et al., 2004; Shvartsman et al., 2003), claudin-3, claudin-4, TROP-1 and CD24 (Santin et al., 2004) and KLK6 (Welsh et al., 2003). Unfortunately, none of these is observed in 100% of EOC samples tested and all are frequently expressed to some degree in the non-malignant conditions, compromising the prospect of any potential assay’s specificity and sensitivity. This is a major problem, given the need for an inexpensive assay as a routine screening procedure. Simply combining two inadequate marker assays tends to corrupt rather than improve the utility of the test (Mills et al., 2001). Furthermore, there is now good evidence some genes are upregulated in a variety of cancers, which may compromise their use as specific biomarkers for EOC (Welsh et al., 2003). More specific assays may be found if the putative EOC marker is alternatively spliced in an EOC specific manner. Such alternative splice forms have been found for the different kallikrein genes (Clements et al., 2004; Borgono et al., 2004) and TADG-12D (Sawasaki et al., 2004). Alternatively, further understanding of the cellular origins of the different types of EOC, combined with knowledge of the mutations leading to EOC and the use of full genome expression profiling arrays, may allow the identification of a bone fide EOC marker with the required specificity and sensitivity. The first attempt to use microarrays to gain insight into EOC aetiology involved comparison of the histopathological subtypes of EOC (Ono et al., 2000; Zheng et al., 2004; Schwartz et al., 2002). Most interestingly, some of the differentially expressed genes agree with independent studies on the cellular morphology of EOC variants. HOX genes, which normally regulate m¨ullerian duct differentiation, are not expressed in normal OSE,

but are expressed in different EOC subtypes according to the pattern of m¨ullerian-like differentiation of these cancers. Ectopic expression of Hoxa9 in tumorigenic mouse OSE cells gives rise to papillary tumours resembling serous EOC (Cheng et al., 2005). In contrast, Hoxa10 and Hoxa11 induced morphogenesis of endometrioid-like and mucinous-like EOC, respectively. Hoxa7 showed no lineage specificity, but promoted the abilities of Hoxa9, Hoxa10 and Hoxa11 to induce differentiation along their respective pathways. Therefore, inappropriate activation of a molecular programme that controls patterning of the reproductive tract could explain the morphologic heterogeneity of EOC and the assumption of m¨ullerian-like features. However, these HOX genes do not appear to be differentially expressed in clear cell, mucinous, and serous EOC expression profiles (Ono et al., 2000; Zheng et al., 2004; Schwartz et al., 2002). Detecting such transcription activators in expression profiles is statistically difficult, because such a low amount of expression is required for them to convey their corresponding biological effects. Indeed, no such histotype-specific genes were identified in the study comparing serous, mucinous, endometrioid and clear cell EOC with normal colon, fallopian tube or endometrium (Marquez et al., 2005). Some EOC expression profiling studies have offered insight into ovarian cancer epidemiology. Although, as discussed, progesterone exposure appears to lower risk of EOC, little is known of its mechanism of action (Ho, 2003; Risch, 1998). A recent study aimed to identify progesterone-regulated genes with potential anti-EOC action (Syed et al., 2005). Transcriptional profiling of normal OSE and EOC cell lines with a cDNA microarray identified genes (1) whose expression was consistently down-regulated in EOC cell lines compared to OSE cell lines, and (2) whose expression was restored in EOC cell lines by progesterone treatment. From the candidates selected, activating transcription factor-3 (ATF-3), caveolin-1, deleted in liver cancer-1 (DLC-1), and non-metastatic clone 23 (NM23-H2) were re-expressed in normal levels in the tumour cell lines in response to progesterone treatment (Syed et al., 2005). 10. Conclusions Despite its huge potential, gene expression profiling of EOC has been confounded by lack of clear understanding of the cellular origin and pathways to EOC. There may be several cells of origin and precursor ovarian states, prior to transformation to malignancy. Although each new array experiment seems to bring up a novel subset of new biomarkers and potential tumour suppressors, results of more recent gene profiling studies are encouraging, because they appear to be starting to identify changes in the expression of gene families and pathways that have occurred during differentiation of the precursor cell to EOC. Our future focus must be to more thoroughly classify the different histotypes by their specific biomarkers and to compare each EOC subtype to a broader range of precursor cells, for example, by using LCM to provide pure reference standard cell populations. The four major hypotheses that arose from the epidemiology of EOC may all be partially supported, but no one hypothesis explains all we now know about ovarian carcinogenesis. A pic-

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Fig. 4. Pathways to EOC are influenced by events at ovulation, including hormonal stimulation and inflammation. Post-ovulatory re-epithelialisation involves OSE (blue cells) cell division and possibly migration, to cover the ovulatory lesion (black arrows). Hyperplasia and transformation of the OSE to adenocarcinoma can occur directly (red arrow top left). The pre-ovulatory luteinising hormone surge induces increased expression of cytokines and invasion of macrophages and monocytes (orange cells), leading to differentiation of follicle cells into luteal cells. Ovulation stimulates formation of invaginations and the formation of inclusion cysts. Cyst cells may differentiate to take on m¨ullerian characteristics under the influence of hormonal or cytokine stimulation and become ciliated (yellow cells) or secretory (tan cells), as shown on the right hand side of the figure. Rete ovarii tubules at the hilus of the ovary, close to the mesothelium to OSE transition (M–E), also contain ciliated and secretory cells and can dilate to form cysts, at least in rodents. It is still not known whether cells in both cyst types can transform to become cancerous. Furthermore, the role of any proposed ovarian stem or progenitor cells (purple cells) in epithelial ovarian carcinogenesis remains to be elucidated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

ture is emerging of at least two pathways; a slower development of high grade EOC via transformation of the cells lining inclusion cysts (Type 1) or a more direct and aggressive development of invasive EOC, directly from the OSE or other epithelial source (Type 2; Fig. 4). The key to determining the origins of EOC is an understanding of the very early changes in these two types of carcinogenic pathway. Animal studies suggest cysts may form from the rete ovarii in some species, so it is possible cysts of different epithelial origin can form in older human ovaries. Studies on the structure and function of the rete ovarii in the adult human are needed before this source of epithelial cell can be totally discounted in EOC. While it is clear incessant ovulation leads to increased rates of invagination and OSE entrapment in the ovarian stroma (Tan et al., 2005), multiple pathways to cyst formation may explain some of the heterogeneity in the large group of serous cystadenocarcinomas. The possibility of ovarian stem cell involvement in EOC also should not be discounted. Further studies are needed to confirm the existence and source of such stem cells and to characterise their responses to ovulation, hormonal stimulation and possible involvement in carcinogenesis. Given the embryonic origin of the rete ovarii, the possibility that this tubule network is a source of such stem cells should also be investigated. Study of inclusion cyst formation under different environmental conditions and endocrine treatments may also help determine the specific contributions of oestrogens, androgens and progestins to inclusion cyst formation and transformation of OSE and cyst cells. Incessant ovulation of transgenic mice, in which genes identified from expression profiling studies are over-expressed or deleted, may allow us to

determine the molecular basis for transformation of cyst cells in Type 1 EOC. Similarly, direct transformation of the OSE in vivo, taking advantage of the bursal capsule surrounding the ovary in the rodent, may be beneficial in defining the molecular basis of Type 2 EOC. The rate of publication on use of whole genome expression profiling in this area has increased rapidly in parallel to access to the technology. Elucidation of the different pathways to EOC and classification of the numerous subtypes of this heterogeneous disease will require a true collaboration of fundamental cell biology and histopathology with molecular genetics. Acknowledgements The authors are indebted to the technical expertise of H. James McQuillan and Melanie Richardson (Department of Anatomy & Structural Biology, University of Otago, New Zealand). They also acknowledge the work of Naomi Armanasco (Department of Anatomy & Structural Biology, University of Otago, New Zealand) on the effects of talc on the mouse ovary. Research on incessant ovulation of mouse ovaries was supported by the New Zealand Lottery Grants Board (Health), the HS & JC Anderson Trust (Otago, New Zealand) and the University of Otago. References Adami, H.O., Hsieh, C.C., Lambe, M., Trichopoulos, D., Leon, D., Persson, I., Ekbom, A., Janson, P.O., 1994. Parity, age at first childbirth, and risk of ovarian cancer. Lancet 344, 1250–1254 (comment).

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