Michael J. Bradaric

Histopathology of ovarian tumors in laying hens: a preclinical model of human ovarian cancer.

The high mortality rate due to ovarian cancer (OVCA) is attributed to the lack of an effective early detection method. Because of the nonspecificity of symptoms at early stage, most of the OVCA cases are detected at late stages. This makes the access to women with early-stage disease problematic and presents a barrier to development and validation of tests for detection of early stage of OVCA in humans. Animal models are used to elucidate disease etiologies and pathogenesis that are difficult to study in humans. Laying hen is the only available animal that develops OVCA spontaneously; however, detailed information on ovarian tumor histology is not available. The goal of this study was to determine the histological features of malignant ovarian tumors in laying hens. A total of 155 young and old (1-5 years of age) laying hens (Gallus domesticus) were selected randomly and evaluated grossly and microscopically for the presence of ovarian tumors. Histological classification of tumors with their stages and grades was determined with reference to those for humans. Similar to humans, all 4 types including serous, endometrioid, mucinous, and clear cell or mixed carcinomas were observed in hen ovarian tumors. Some early neoplastic as well as putative ovarian lesions were also observed. Similarities in histology, metastasis, and stages of hen OVCA to those of humans demonstrate the feasibility of the hen model for additional delineation of the mechanism underlying ovarian carcinogenesis, preclinical testing of new agents for the prevention, and therapy of this disease.

Differential expression of aldehyde dehydrogenase 1a1 (ALDH1) in normal ovary and serous ovarian tumors.

BackgroundWe showed there are specific ALDH1 autoantibodies in ovarian autoimmune disease and ovarian cancer, suggesting a role for ALDH1 in ovarian pathology. However, there is little information on the ovarian expression of ALDH1. Therefore, we compared ALDH1 expression in normal ovary and benign and malignant ovarian tumors to determine if ALDH1 expression is altered in ovarian cancer. Since there is also recent interest in ALDH1 as a cancer stem cell (CSC) marker, we assessed co-expression of ALDH1 with CSC markers in order to determine if ALDH1 is a potential CSC marker in ovarian cancer.MethodsmRNA and protein expression were compared in normal human ovary and serous ovarian tumors using quantitative Reverse-Transcriptase PCR, Western blot (WB) and semi-quantitative immunohistochemistry (IHC). ALDH1 enzyme activity was confirmed in primary ovarian cells by flow cytometry (FC) using ALDEFLUOR assay.ResultsALDH1 mRNA expression was significantly reduced (p < 0.01; n = 5) in malignant tumors compared to normal ovaries and benign tumors. The proportion of ALDH1+ cells was significantly lower in malignant tumors (17.1 ± 7.61%; n = 5) compared to normal ovaries (37.4 ± 5.4%; p < 0.01; n = 5) and benign tumors (31.03 ± 6.68%; p < 0.05; n = 5). ALDH1+ cells occurred in the stroma and surface epithelium in normal ovary and benign tumors, although surface epithelial expression varied more in benign tumors. Localization of ALDH1 was heterogeneous in malignant tumor cells and little ALDH1 expression occurred in poorly differentiated malignant tumors. In benign tumors the distribution of ALDH1 had features of both normal ovary and malignant tumors. ALDH1 protein expression assessed by IHC, WB and FC was positively correlated (p < 0.01). ALDH1 did not appear to be co-expressed with the CSC markers CD44, CD117 and CD133 by IHC.ConclusionsTotal ALDH1 expression is significantly reduced in malignant ovarian tumors while it is relatively unchanged in benign tumors compared to normal ovary. Thus, ALDH1 expression in the ovary does not appear to be similar to breast, lung or colon cancer suggesting possible functional differences in these cancers.SignificanceThese observations suggest that reduced ALDH1 expression is associated with malignant transformation in ovarian cancer and provides a basis for further study of the mechanism of ALDH1 in this process.

Detection of Tumor-Associated Neoangiogenesis by Doppler Ultrasonography During Early-Stage Ovarian Cancer in Laying Hens A Preclinical Model of Human Spontaneous Ovarian Cancer

Objective. Tumor‐associated neoangiogenesis (TAN) is one of the earliest events in ovarian tumor growth and represents a potential target for early detection of ovarian cancer (OVCA). Because it is difficult to identify patients with early‐stage OVCA, the goal of this study was to explore a spontaneous animal model of in vivo ovarian TAN associated with early‐stage OVCA detectable by Doppler ultrasonography (DUS). Methods. White Leghorn laying hens were scanned transvaginally at 15‐week intervals up to 45 weeks. Gray scale ovarian morphologic characteristics and Doppler indices were recorded. Hens were euthanized at diagnosis for ultrasonographic morphologic/vascular abnormalities or at the end of the study (those that remained normal). Ovarian morphologic and histologic characteristics were evaluated. Vascular endothelial growth factor (VEGF) and αvβ3‐integrin expression was assessed by immunohistochemical analysis. Doppler ultrasonographic observations were compared with histologic and immunohisto‐chemical findings to determine the ability of DUS to detect ovarian TAN. Results. Significant changes in ovarian blood flow parameters were observed during transformation from normal to tumor development in the ovary (P < .05). Tumor‐related changes in ovarian vascularity were identified by DUS before the tumor became detectable by gray scale imaging. Increased expression of VEGF and αvβ3‐integrins was associated with tumor development. Ovarian TAN preceded tumor progression in hens. Conclusions. The results suggest that ovarian TAN may be an effective target for the detection of early‐stage OVCA. The laying hen may also be useful for studying the detection and inhibition of ovarian TAN using various means, including the efficacy of contrast agents, targeted molecular imaging, and antiangiogenic therapies.

Detection of Ovarian Tumors in Chicken by Sonography A Step Toward Early Diagnosis in Humans

Animal models of spontaneous ovarian cancer are important for understanding early tumor development. Ovarian imaging may play an important role in following changes in tumor development. Laying hens are the only animals that develop spontaneous ovarian cancer similar to humans. The aim of this study was to determine the feasibility of detecting ovarian tumors in laying hens using sonography.

Anti‐tumor and Anti‐ovarian Autoantibodies in Women with Ovarian Cancer

There is a lack of validated marker(s) for the diagnosis of early‐stage ovarian cancer (OVCA). The objective was to determine if women with OVCA had antibodies, to assess their potential as markers of ovarian cancer. The secondary objective was to compare the prevalence of antibodies to proteins from normal ovary and ovarian tumors to determine if antibodies primarily recognize tumor antigens, as many antigens are common to tumor and normal ovary.

Prevalence of antitumor antibodies in laying hen model of human ovarian cancer.

Antitumor antibodies are associated with tumors in human cancers. There is relatively little information on the timing and progression of antibody response to tumors. The objective of the study was to determine if spontaneous ovarian cancer in the egg-laying hen is associated with antitumor antibodies. Antibodies were detected by immunoassay and immunoblotting using proteins from normal ovary and ovarian tumors. Candidate antigens were identified by mass spectrometry of immunoreactive spots cut from 2-dimensional gels and Western blot. Antitumor (serum reacting against tumor ovarian extract) and antiovarian (serum reacting against normal ovarian extract) antibodies were significantly associated with ovarian cancer (67%; P ≤ 0.001) compared with normal control hens. Hens with abnormal histology but no gross tumor had antitumor antibodies (63%; P ≤ 0.025) but not antiovarian antibodies. There were common as well as different immunoreactions against normal ovary and homologous and heterologous tumor proteins in 2-dimensional Western blots. The candidate antigens included those commonly associated with human cancers and other diseases such as vimentin, apolipoprotein A1, Annexinn II, enolase, DJ-1, and so on. The results suggest that antitumor antibodies are associated with ovarian cancer in hens, similar to human ovarian cancer. The egg-laying hen may be a model for understanding the antitumor humoral immune response, particularly at early tumor stages that are not readily accessible in human ovarian cancer.

Statin therapy is associated with improved survival in patients with non-serous-papillary epithelial ovarian cancer: a retrospective cohort analysis.

Aim To determine whether statin use is associated with improved epithelial ovarian cancer (OvCa) survival. Methods This is a single-institution retrospective cohort review of patients treated for OvCa between 1992 and 2013. Inclusion criteria were International Federation of Gynecology and Obstetrics (FIGO) stage I–IV OvCa. The primary exposures analyzed were hyperlipidemia and statin use. The primary outcomes were progression-free survival (PFS) and disease-specific survival (DSS). Results 442 patients met inclusion criteria. The cohort was divided into three groups: patients with hyperlipidemia who used statins (n = 68), patients with hyperlipidemia who did not use statins (n = 28), and patients without hyperlipidemia (n = 346). OvCa outcomes were evaluated. When we analyzed the entire cohort, we found no significant differences in PFS or DSS among the groups. The median PFS for hyperlipidemics using statins, hyperlipidemics not using statins, and non-hyperlipidemics was 21.7, 13.6, and 14.7 months, respectively (p = 0.69). Median DSS for hyperlipidemics using statins, hyperlipidemics not using statins, and non-hyperlipidemics was 44.2, 75.7, and 41.5 months, respectively (p = 0.43). These findings did not change after controlling for confounders. However, a secondary analysis revealed that, among patients with non-serous-papillary subtypes of OvCa, statin use was associated with a decrease in hazards of both disease recurrence (adjusted HR = 0.23, p = 0.02) and disease-specific death (adjusted HR = 0.23, p = 0.04). To augment the findings in the retrospective cohort, the histology-specific effects of statins were also evaluated in vitro using proliferation assays. Here, statin treatment of cell lines resulted in a variable level of cytotoxicity. Conclusion Statin use among patients with non-serous-papillary OvCa was associated with improvement in both PFS and DSS.

Contrast-Enhanced Sonography Depicts Spontaneous Ovarian Cancer at Early Stages in a Preclinical Animal Model

Our goal was to examine the feasibility of using laying hens, a preclinical model of human spontaneous ovarian cancer, in determining the kinetics of an ultrasound contrast agent indicative of ovarian tumor‐associated neoangiogenesis in early‐stage ovarian cancer.

Immune Cells in the Normal Ovary and Spontaneous Ovarian Tumors in the Laying Hen (Gallus domesticus) Model of Human Ovarian Cancer

Background Spontaneous ovarian cancer in chickens resembles human tumors both histologically and biochemically. The goal was to determine if there are differences in lymphocyte content between normal ovaries and ovarian tumors in chickens as a basis for further studies to understand the role of immunity in human ovarian cancer progression. Methods Hens were selected using grey scale and color Doppler ultrasound to determine if they had normal or tumor morphology. Cells were isolated from ovaries (n = 6 hens) and lymphocyte numbers were determined by flow cytometry using antibodies to avian CD4 and CD8 T and B (Bu1a) cells. Ovarian sections from another set of hens (n = 26) were assessed to verify tumor type and stage and to count CD4, CD8 and Bu1a immunostained cells by morphometric analysis. Results T and B cells were more numerous in ovarian tumors than in normal ovaries by flow cytometry and immunohistochemistry. There were less CD4+ cells than CD8+ and Bu1a+ cells in normal ovaries or ovarian tumors. CD8+ cells were the dominant T cell sub-type in both ovarian stroma and in ovarian follicles compared to CD4+ cells. Bu1a+ cells were consistently found in the stroma of normal ovaries and ovarian tumors but were not associated with follicles. The number of immune cells was highest in late stage serous tumors compared to endometrioid and mucinous tumors. Conclusions The results suggest that similar to human ovarian cancer there are comparatively more immune cells in chicken ovarian tumors than in normal ovaries, and the highest immune cell content occurs in serous tumors. Thus, this study establishes a foundation for further study of tumor immune responses in a spontaneous model of ovarian cancer which will facilitate studies of the role of immunity in early ovarian cancer progression and use of the hen in pre-clinical vaccine trials.

Dietary Supplementation of Ashwagandha (Withania somnifera, Dunal) Enhances NK Cell Function in Ovarian Tumors in the Laying Hen Model of Spontaneous Ovarian Cancer

Ovarian cancer (OVCA) disseminates in a distinct pattern through peritoneal metastasis and little is known about the immunosuppression in the tumor microenvironment. Our goal was to determine changes in NK cell population during OVCA development and the effects of Ashwagandha (Withania somnifera, Dunal) supplementation on NK cell localization in laying hens with OVCA.