**5. Histological characteristics of endometriosis-associated malignancies**

Clear cell carcinoma (Fig. 2) and endometrioid adenocarcinoma are well-known histological subtypes in ovarian cancer associated with endometriosis (Fukunaga, 1997; Heaps, 1990; Modesitt, 2002; Ogawa, 2000; Yoshikawa, 2000). Endometrioid adenocarcinoma is the most frequently observed phenotype in western countries (Heaps, 1990; Modesitt, 2002); however, clear cell carcinoma predominates in the Japanese cases (Ogawa, 2000; Yoshikawa, 2000). Veras et al. recently subdivided clear cell carcinoma into 3 groups (cystic, adenofibromatous, and indeterminate clear cell carcinoma) to further reveal the association between endometriosis and cystic clear cell carcinoma subtypes (Veras, 2009). Endometrioid adenocarcinomas arising in endometriotic lesions are often Grade 1 at presentation (Horiuchi, 2003), mostly showing typical morphology with various degrees of squamous differentiation (Heaps, 1990; Staats, 2007), similar to endometrioid adenocarcinoma without endometriosis. Sarcomas are the second and third most frequent endometriosis-associated

tumor suppressor genes, promoter methylation of oncogenes, nor oncogenic mutations of known tumor-related genes was frequently observed in the majority of the cases, further denying the neoplastic theory (Prowse, 2005; Vestergaard AL, 2011). In contrast with these results, a third approach (fluorescent in situ hybridization [FISH]) used to investigate chromosomal aberrations in endometriosis samples revealed a significantly elevated proportion of aneusomic (monosomic > trisomic) cells in endometriosis in multiple groups (Koerner, 2006) (Bischoff, 2002). However, both endometriosis tissue and normal endometrium also contain a certain proportion of aneusomic cells (Koerner, 2006), and telomerase expression, telomere elongation, higher expression of DNA replication markers and lower expression of DNA damage response markers are all observed in endometriosis tissue, but not in normal endometrium (Hapangama, 2008; Hapangama, 2009). Thus, it may be reasonable to conclude that although endometriosis is generally considered non-neoplastic, the relative rates of abnormal cells are higher in endometriosis

In this case, then, which cells are premalignant? Is there a focal area representing the precancerous state of endometriosis that is morphologically distinguishable from other, presumably benign, areas? "Atypical endometriosis" is the term used to describe this state, which has been found in cases of extraovarian and ovarian cancer as atypical epithelium showing hyperchromatism and stratification continuous with the malignant tumor (Brooks&Wheeler, 1977; Lagrenade&Silverberg, 1988). Fukunaga et al. found atypical endometriosis in 61% of endometriosis-associated ovarian cancers, in contrast with 1.7% of benign endometriosis samples (Fukunaga, 1997). Immunohistochemical markers distinguishing atypical endometriosis from benign endometriosis have not been fully established, but staining patterns of Ki67, Bcl-2, and p53 have been reported as useful markers (Nezhat, 2002; Ogawa, 2000). Extraovarian endometriosis may also show atypical changes. Hyperplastic changes, including atypical hyperplasia and malignant changes, were observed in more than half of the adenomyosis cases associated with endometrioid adenocarcinoma arising from the endometrium (Jacques&Lawrence, 1990; Kucera, 2011), and histologically atypical hyperplasia has been reported in some cases of gastrointestinal

**5. Histological characteristics of endometriosis-associated malignancies** 

Clear cell carcinoma (Fig. 2) and endometrioid adenocarcinoma are well-known histological subtypes in ovarian cancer associated with endometriosis (Fukunaga, 1997; Heaps, 1990; Modesitt, 2002; Ogawa, 2000; Yoshikawa, 2000). Endometrioid adenocarcinoma is the most frequently observed phenotype in western countries (Heaps, 1990; Modesitt, 2002); however, clear cell carcinoma predominates in the Japanese cases (Ogawa, 2000; Yoshikawa, 2000). Veras et al. recently subdivided clear cell carcinoma into 3 groups (cystic, adenofibromatous, and indeterminate clear cell carcinoma) to further reveal the association between endometriosis and cystic clear cell carcinoma subtypes (Veras, 2009). Endometrioid adenocarcinomas arising in endometriotic lesions are often Grade 1 at presentation (Horiuchi, 2003), mostly showing typical morphology with various degrees of squamous differentiation (Heaps, 1990; Staats, 2007), similar to endometrioid adenocarcinoma without endometriosis. Sarcomas are the second and third most frequent endometriosis-associated

than in normal endometrium.

endometriosis (Yantiss, 2000).

extraovarian and ovarian tumors, respectively. Adenosarcoma and endometrial stromal sarcoma are the major histological types of sarcomas (Baiocchi, 1990; Heaps, 1990; Slavin, 2000). At least partially, differences in the incidences of tumor types (carcinoma versus sarcoma) depend on the tumor site, and further studies are needed to elucidate this mechanism. Other rare malignant tumors, such as squamous cell carcinoma, malignant mesodermal mixed tumor, and yolk sac tumor, are also reported to develop from endometriosis (Irving, 2011). Although its incidence is very low compared with endometrioid adenocarcinoma or clear cell carcinoma, serous adenocarcinoma has also been associated with endometriosis (Fukunaga, 1997; Modesitt, 2002; Yoshikawa, 2000). Much more rarely, mucinous carcinomas with unusual morphology resembling Mullerian mucinous borderline tumors have also been reported in association with endometriosis (Lee&Nucci, 2003).

Fig. 2. A. Clear cell carcinoma (left) arising in a endometriotic cyst. B. Hemosiderin deposition (arrows) is observed in the stroma of clear cell carcinoma.

#### **6. Genetic abnormalities and phenotypes of endometriosis-associated ovarian cancer**

Genetic mutations specifically associated with ovarian cancer subtypes have been reported (reviewed by (Kurman&Shih, 2011)). Focusing on endometrioid adenocarcinomas, genetic mutations of K-ras, p53, PTEN, beta-catenin, and ATR have been reported ( Mizuuchi, 1992; Milner, 1993; Palacios&Gamallo, 1998; Tashiro, 1997; Zighelboim, 2009). Mouse models of endometrioid adenocarcinoma have been reported, either with oncogenic K-ras and conditional PTEN deletion (Dinulescu, 2005) or dysfunction of both the Wnt/beta-catenin and PI3CA/PTEN pathways (Wu, 2007). However, specific genetic alterations of clear cell carcinoma were mostly unknown. Recently, a frequently activated mutation of the PI3CA gene was observed in clear cell carcinoma samples (Kuo, 2009). Most recently, several studies based on novel sequencing technology have elucidated that a significant proportion of clear cell carcinomas harbor a mutation of the ARID1A gene, which encodes the chromatin-remodeling complex protein BAF250A (Jones, 2010; Wiegand, 2010). ARID1A mutation and the consequent loss of BAF250A expression were found not only in clear cell carcinoma samples, but also in endometrioid adenocarcinomas, especially high-grade types

Endometriosis-Associated Ovarian Cancer: The Role of Oxidative Stress 317

histological subtypes using a small number of biomarkers has been applied to ovarian cancers. A tissue microarray-based analysis selected 21 markers, including CA125, estrogen receptor (ER), insulin-like growth factor 2 (IGF2), Ki-67, p21, p53, progesterone receptor (PGR), and Wilms tumor 1 (WT1), to distinguish histological subtypes; however, only three of the 21 markers could predict outcomes in only high-grade serous carcinoma patients (Koebel, 2008). More recently, however, Kalloger et al. succeeded in reproductively diagnosing five major subtypes of ovarian cancers (high-grade serous, clear cell, endometrioid, mucinous, and low-grade serous) using only nine markers: p16, DKK1 (a Wnt antagonist), HNF-1β, MDM2, PGR, trefoil factor 3 (TFF3), p53, vimentin, and WT1 (Kalloger, 2011). Immunohistochemical analysis of 155 cases by De Lair et al demonstrated that 89% of clear cell carcinoma had HNF-1β positive, ER, PGR, and WT1

Clear cell adenocarcinoma is known to be associated with chemoresistancy and a poor prognosis (Itamochi, 2008). However, most reports analyzing the prognosis of endometriosis-associated ovarian carcinomas (including mostly endometrioid adenocarcinoma and few clear cell carcinoma samples) have shown that endometriosisassociated ovarian carcinomas presented at younger ages, in lower grades and stages, and had significantly better overall survival compared with age-matched controls without endometriosis (Erzen, 2001; Kumar, 2011; Melin, 2011 ; Orezzoli, 2008). However, recent studies from various countries indicate that clear cell carcinomas consist of heterogenous tumors with gene alterations, such as HER2 or Met gene amplification (Tan, 2011 ; Yamamoto, 2011; Yamashita, 2011). Therefore, clear cell carcinomas as a subtype are considered to have a worse prognosis than endometrioid adenocarcinomas, especially in Asian cases (Lee, 2011). Recently, the first international symposium of ovarian clear cell carcinoma concluded that although patients with low-stage clear cell carcinoma had a better prognosis than matched controls with high-grade serous carcinoma, high-stage clear cell carcinoma cases had the worst prognosis (Anglesio, 2010). Thus, alternative therapy, such as molecular targeted therapy, should be applied to these aggressive tumors, and a further understanding of the basic biology of the endometriosis-cancer progression, especially the role of oxidative stress, is necessary to prevent carcinogenesis in endometriosis patients

We have reviewed the literature on endometriosis-associated ovarian cancer. Further

Anglesio, M. S., Carey, M. S., Koebel, M., MacKay, H. and Huntsman, D. G. (2010). Clear cell

Aris, A. (2010). Endometriosis-associated ovarian cancer: A ten-year cohort study of women living in the Estrie Region of Quebec, Canada. *Journal of Ovarian Research* 3, 2.

carcinoma of the ovary: A report from the first Ovarian Clear Cell Symposium,

studies are awaited to clarify the exact role of oxidative stress in carcinogenesis.

June 24th, 2010. *Gynecologic Oncology* 121, 407-415.

negative phenotype (DeLair, 2011).

(Aris, 2010).

**8. Conclusion** 

**9. References** 

**7. Prognosis of endometriosis-associated ovarian cancer** 

(Wiegand 2010; Wiegand, 2011). Whether ARID1A mutation is an early or late event in endometriosis-associated ovarian cancers related to atypical endometriosis remains to be elucidated. Alterations of other genes, such as p53, p16, and PTEN, have been detected in a low percentage of endometriotic lesions (Martini, 2002; Nezhat, 2008). hMLH, a DNA mismatch repair gene, is another candidate for the malignant transformation of endometriosis (Nyiraneza, 2010 ; Ren F, 2011). hMLH is the causal gene of Lynch syndrome, in which the risk of developing endometrial and ovarian cancers is significantly increased (Schmeler&Lu, 2008). K-ras may also be important because mutated K-ras promotes endometriosis in a mouse model, suggesting that K-ras mutation may be an early event in the carcinogenesis of endometriosis-associated cancers (Cheng, 2011). Finally, a singlenucleotide polymorphism in the intron of ANRIL, a non-coding RNA that regulates p16 expression, has been recently reported to have a strong association with endometriosis (Uno, 2010). The molecular steps from endometriosis development to carcinogenesis remain to be further clarified.

Recent studies have proposed classifying ovarian cancers into two categories: Type I tumors, which rarely harbor the p53 mutation and have an indolent clinical course, and Type II tumors, which feature the p53 mutation and are aggressive (Kurman&Shih, 2010). Within endometriosis-associated ovarian cancers, low-grade endometrioid adenocarcinoma and clear cell carcinomas are considered Type I, while high-grade endometrioid adenocarcinoma is included in the Type II category. However, p53 mutations are detected in both low- and high-grade endometriosis-associated ovarian endometrioid adenocarcinomas (Okuda, 2003), and PI3CA, PPP2R1A, and K-ras mutations are commonly detected in both endometrioid adenocarcinoma and clear cell carcinoma (Campbell, 2004; Jones, 2010 ; Kuo, 2009; McConechy, 2011 ; Mizuuchi, 1992). Recent evidence indicates that ovarian cancers arise from different cell lineages, such as preexisting cystadenomas, ectopic endometrium in endometriotic lesions, and epithelial cells of the Fallopian tubes (Bell, 2005; Kurman&Shih, 2011). Thus, it may be an oversimplification to divide all ovarian cancers into two groups. It may more accurate to categorize endometriosis-associated cancers into the same group, regardless of the histological subtype or tumor grade.

Numerous studies of expression microarray analyses have been published. Cytokines and chemokines, such as interleukin-1 and its downstream factor cyclooxygenase (COX)-2, interleukin-8, TNF-α and its downstream VEGF, TGF- α, and interleukin-6 have been reported to be involved in endometriosis and endometriosis-associated carcinoma (reviewed by (Nezhat, 2008)). An interesting study by Banz et al. revealed that SICA2, CCL14, and TDGF1 were specifically upregulated in both endometriosis samples and endometriosis-associated endometrioid adenocarcinomas, in contrast with serous adenocarcinomas or normal ovarian tissues (Banz, 2010). Another microarray study focusing on endometriosis-associated clear cell carcinoma showed upregulation of hepatocyte nuclear factor (HNF)-1β, versican, and other markers related to oxidative stress (Yamaguchi, 2010). HNF-1β is a transcription factor, involved in the regulation of glucose homeostasis and glycogen accumulation, normally expressed in the liver and other organs, which is assumed to have some role in the pathogenesis of clear cell carcinoma of the ovary (Kobayashi, 2009). Recently, a novel attempt to classify histological subtypes using a small number of biomarkers has been applied to ovarian cancers. A tissue microarray-based analysis selected 21 markers, including CA125, estrogen receptor (ER), insulin-like growth factor 2 (IGF2), Ki-67, p21, p53, progesterone receptor (PGR), and Wilms tumor 1 (WT1), to distinguish histological subtypes; however, only three of the 21 markers could predict outcomes in only high-grade serous carcinoma patients (Koebel, 2008). More recently, however, Kalloger et al. succeeded in reproductively diagnosing five major subtypes of ovarian cancers (high-grade serous, clear cell, endometrioid, mucinous, and low-grade serous) using only nine markers: p16, DKK1 (a Wnt antagonist), HNF-1β, MDM2, PGR, trefoil factor 3 (TFF3), p53, vimentin, and WT1 (Kalloger, 2011). Immunohistochemical analysis of 155 cases by De Lair et al demonstrated that 89% of clear cell carcinoma had HNF-1β positive, ER, PGR, and WT1 negative phenotype (DeLair, 2011).
