Using the Domestic Hen as a Model for Studying Ovarian Cancer
The Ribbon

P. A. Johnson* and J.R. Giles

Department of Animal Science, Cornell University

Since BCERF joined Cornell's College of Veterinary Medicine in 2004, readers of this publication, visitors to our web site, and participants in the Regional Cancer and Environment Forums are hopefully becoming more aware of the way in which the study of spontaneously arising cancers in wildlife and domesticated animals are increasing our understanding of human cancer. Below we highlight some very promising research, taking place within Cornell's Department of Animal Science, addressing ovarian cancer. Collaborations are currently underway between this lab and investigators at Roswell Park Cancer Institute.

This article is adapted with permission from the Journal of Poultry Science, originally published as "Use of Genetic Strains of Chickens in Studies of Ovarian Cancer," Volume 85, Number 2, February 2006, pp. 246-250.

Background on human ovarian cancer

Ovarian cancer is the fifth leading cause of death from all cancers among women and it is a leading cause of gynecological malignancies. It is estimated that nearly 25,000 new cases of ovarian cancer will be diagnosed this year and more than half that many women die from this cancer each year (NCI, http://seer.cancer.gov/csr/1975_2002/results_single/sect_01_table.01.pdf). Ovarian cancer occurs with an approximate incidence of 1 out of 57 women. The estimated percentage of survival to five years is strongly influenced by the stage at which the cancer is diagnosed. The five-year survival for early stage tumors (stages I and II) is approximately 80-90 percent while that for later stage tumors (stages III and IV) is much less at 5-40 percent (Chi and Hoskins, 2000). Unfortunately, a large study (Pettersson et al., 1991) has indicated that a majority (approximately 65 percent) of tumors are not discovered until stages III or IV. This is likely related to the few symptoms experienced by women with ovarian cancer at early stages.

Ovarian tumors are believed to arise from several sites on the ovary. Most cases of ovarian cancer are termed "epithelial" and are believed to arise from the surface epithelium, or, the single layer of tissue covering the surface of the ovary. Seven to ten percent of cases are termed non-epithelial and arise from the germ cells or stroma of the ovary. Family history, including genetic mutations such as in the BRCA genes (Auersperg et al., 2001), accounts for only about five percent of cases, while most cases are sporadic. The strongest risk factor for ovarian cancer is age, where the risk is low for young women and increases throughout reproductive life to plateau at about age 55 (Banks, 2000).

There are other factors that have been associated with alteration of risk for ovarian cancer. An important factor is pregnancy. A full-term pregnancy is associated with a risk reduction of approximately 40 percent (Banks, 2000). Moreover, each subsequent pregnancy confers an additional 10-15 percent reduction of risk. Use of the oral contraceptive pill for three years is associated with a 40 percent reduction of risk for ovarian cancer. Each additional year of use reduces the risk by five to ten percent (reviewed by Banks, 2000). Fathalla (1971) proposed that frequent ovulation contributes to increased risk for ovarian cancer. His hypothesis was termed "the incessant ovulation hypothesis." He proposed that repeated rupture and repair of the ovarian surface epithelium provides the opportunity for genetic aberrations. The single cell layer surrounding the ovary must be repaired after each ovulation. It is possible that areas of the surface epithelium become detached from the surface and become part of so-called inclusion cysts (reviewed by Auersperg, 2001). These "inclusion bodies" may provide an abnormal environment for the epithelial cells and are implicated in the origin of ovarian cancer. One study has indicated that women with ovarian cancer have been observed to have an increased incidence of inclusion cysts in the other ovary (Salazar et al., 1996). The incessant ovulation hypothesis is supported by the epidemiological data relating to pregnancy and oral contraceptive use. It is also possible that endocrine factors are involved in the genesis or progression of ovarian cancer.

Why is the domestic hen a good model for human ovarian cancer?

Most animals do not spontaneously develop ovarian cancer and this has made the study of the tumors difficult (MacLachlan, 1987). A variety of rodent models (Orsulic et al., 2002; Connolly et al., 2003) have been utilized as well as cell lines from human tumors (Langdon and Lawrie, 2000) and normal ovarian surface epithelial cells (Auersperg and Maines-Bandiera, 2000). These models have been useful but study of the origin and development of early tumors is limited. Generally, among domestic animals, the desired state is pregnancy and/or lactation and most wild animals are pregnant, lactating or seasonally sexually inactive. These physiological states are not associated with frequent ovulation or ovarian cancer. The one model that does exhibit ovarian cancer with a high incidence is the domestic hen (Campbell, 1951; Wilson, 1958; Fredrickson, 1987). This observation, along with the fact that the hen is a persistent ovulator, makes the hen a good model for the disease. Many commercial strains of laying hens ovulate almost daily through one or two years of egg production. This is similar to the pattern experienced by many contemporary women who ovulate monthly for 10-20 years, have one or two closely spaced pregnancies, and then resume ovulation for 10-20 more years.

Fredrickson (1987) conducted a three and one half-year study in which he evaluated the incidence of reproductive tract tumors in 466 White Leghorn hens ranging from two to seven years of age. He found that 24 percent of all hens developed age-dependent malignant ovarian tumors. He also observed that these tumors were uncommon in hens less than two years of age and that ovulation rate was not associated with incidence. Hormonal imbalance did not appear to be a factor although the hormone levels were very variable.

Findings from the research analyzing characteristics of the chicken ovarian tumors suggest several features in common with the most typical type found in women. For example, one study has shown that hen ovarian tumors are cross-reactive with many antibodies used to detect several antigens in human ovarian cancers (Rodriguez-Burford et al., 2001). In our research we found that progesterone receptor is expressed in the cells lining the glands of hen ovarian cancers similar to women. We have also found that hen ovarian cancers are positive for expression of the oviductal protein, ovalbumin (Giles et al., 2004). The finding of ovalbumin expression suggested that hen ovarian cancer was similar to the most common type of ovarian cancer found in women which has oviduct-like characteristics.

Variations in ovarian cancer rates between different genetic strains.

Studies have shown genetic differences in susceptibility to ovarian tumors among selected lines of hens with one flock having about a five-fold greater tumor incidence than another flock (Fredrickson, 1987). Here at Cornell, two closed strains of White Leghorn hens (Cornell C and K) have been maintained since 1935 and 1936, respectively (Cole and Hutt, 1973). These strains were derived from a similar genetic background and selected for disease resistance combined with selection for other important production traits. In recent years, they have been maintained with random breeding. Cole and Hutt (1973) observed differences in rate of reproductive cancer between the strains. In two separate studies, we found that two year-old C strain of hens had a significantly (p< 0.02) increased rate of ovarian cancer compared to two year-old hens of the K strain. In addition, we found that C strain hens had an overall significantly higher incidence (p < 0.05) of ovarian cancer compared to the K strain hens. A genetic association in human epithelial ovarian cancer is well documented, however, this accounts for only a small proportion of those with the cancer. Most cases of epithelial ovarian cancers occur in women with no family history of the disease.

The main thrust of our laboratory is reproductive endocrinology and therefore, our interest relates to possible hormonal correlates of ovarian cancer. Estrogen-only replacement therapy has been indicated as a risk factor for ovarian cancer (Lacey et al., 2002) and this has led us to examine plasma estradiol in our two strains of hens. Preliminary analysis showed that basal plasma estradiol was elevated in the C strain of hens as compared to the K strain hens (Davignon and Johnson, unpublished). Furthermore, plasma estradiol was higher at all ages examined (one, two and three years of age) in C strain hens as compared to K strain hens, suggesting that the C strain may be exposed to chronic, higher levels of estradiol. Egg production, as a reflection of ovulation rate, and plasma progesterone, were not different between the strains. The biological basis for the difference in plasma estradiol may be due to a significantly larger ovary in C strain hens (p< 0.02), with the result of more small follicles capable of estradiol production (Robinson and Etches, 1986).

Another hormone that has been implicated in the development or progression of ovarian tumors is the gonadal hormone inhibin. Matzuk and co-workers (Matzuk et al., 1992) have demonstrated that inhibin may be a tumor suppressor factor in mice. We hypothesized that inhibin may be expressed at a lower level in the hens more prone to ovarian cancer (C strain) as compared to the K strain hens. For this reason, we examined inhibin in the plasma and messenger RNA expression in the granulosa layer of the C and K strain hens. In two separate trials, plasma immunoreactive inhibin was significantly lower (P < 0.02 and P < 0.05 for trial 1 and 2, respectively) in C strain hens compared to K strain hens. We found that inhibin messenger RNA was expressed at a lower level (P < 0.02) in the C strain as compared to the K strain. The role of inhibin in ovarian cancer in the hen warrants further study.

Prostaglandins have been implicated in a variety of cancers and the enzymes responsible for their production (COX-1 and COX-2) have been targeted as potential therapies. In many tumors such as colon and lung, COX-2 has been implicated. Recent studies suggest that COX-1 may be selectively increased in human ovarian cancer (Gupta, et al., 2003) and this has also been found to be the case in the hen (Urick and Johnson, in press). These findings may implicate COX-1 inhibitors (such as non-steroidal anti-inflammatory drugs) as suitable targets for the prevention or treatment of ovarian cancer.

In summary

The etiology of ovarian cancer in humans is poorly understood, in part from a lack of animal models. One animal that has been shown to spontaneously develop the disease is the domestic hen. Similar to women, the incidence of ovarian tumors in hens increases with age and exhibits metastases to similar abdominal tissues. A genetic component has been shown in hens with the incidence influenced by strain. Ovarian tumors are more common in the C strain of White Leghorn hens compared to the K strain. Interestingly, C strain hens also have greater plasma estradiol levels and larger ovaries than K strain hens. Furthermore, blood plasma inhibin levels were reduced for C strain hens compared to K strain hens. Finally, ovarian tumors in hens show increased expression of COX-1 similar to humans, suggesting a possible therapy and further validating the hen as a good model for humans. In conclusion, analysis of genetic differences in hens and the relationship of these factors to the incidence of ovarian cancer may be very helpful in learning more about the etiology of the disease in the chicken and hopefully, can be applied to this very lethal disease in humans.

Ovarian cancer and immunotherapy: Dr. Kunle Odunsi's work at Roswell Park Cancer Institute

There is growing evidence for a link between the immune system and the control of cancer. Support for this link comes from observations that the immune system can protect against the development of spontaneous and chemically induced tumors in laboratory research. Further, a large number of targets for immune recognition of human cancer have been identified and characterized. Results of a large study published in Proceedings of the National Academy of Sciences (Sato et al., 2005) provide further evidence for the role of the immune system in controlling cancer. The research team, led by Dr. Kunle Odunsi, Associate Professor, Department of Gynecologic Oncology at Roswell Park Cancer Institute (RPCI), examined the precise location of subpopulations of immune cells [tumor infiltrating lymphocytes (TILs)] in 117 RPCI patients with epithelial ovarian cancer (EOC) to determine the interrelationship between subpopulations of TILs and overall survival. The results indicate that the presence of high numbers of killer immune cells (CD8+ TILs) is associated with favorable prognosis in ovarian cancer patients.

Based on these observations, Dr. Odunsi is currently conducting studies aimed at inducing or augmenting CD8+ immune cells, in order to improve outcome for ovarian cancer patients. The studies are testing the safety and effectiveness of a vaccine treatment derived from a protein, called NY-ESO-1, as immunotherapy for patients with ovarian, fallopian tube or primary peritoneal cancer.

This work is supported by a Cancer Vaccine Collaborative Grant from the Cancer Research Institute (CRI) and the Ludwig Institute for Cancer Research, and an Anna-Marie Kellen Clinical Investigator Award from the CRI to Dr. Kunle Odunsi.

Acknowledgments

We thank C. Brooks, D. Davignon, and J. Jagne of Cornell University, Ithaca, NY and L. Olson of Washington University, St. Louis, MO for their contributions to the work described in this manuscript. We are also grateful H. Shivaprasad at Univ. of California at Davis, Davis, CA for his help during the work. Finally, we thank the workers at the Cornell Poultry Farm for their care of the genetic strains of hens. This work has been supported by the Department of Defense Ovarian Cancer Research Program and core support from CALS and the Department of Animal Science at Cornell University.

*Address correspondence to:
paj1@cornell.edu
Dr. P.A. Johnson, 202 Morrison Hall, Cornell University, Ithaca, NY 14853.
Phone: 607-255-3077; FAX: 607-255-9829.

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Using the Domestic Hen as a Model for Studying Ovarian Cancer The Ribbon

Using the Domestic Hen as a Model for Studying Ovarian Cancer
The Ribbon