Paraffin-embedded Ovarian Tissue Research Articles

Human ovarian aging is characterized by oxidative damage and mitochondrial dysfunction.

Are human ovarian aging and the age-related female fertility decline caused by oxidative stress and mitochondrial dysfunction in oocytes? We found oxidative damage in oocytes of advanced maternal age, even at the primordial follicle stage, and confirmed mitochondrial dysfunction in such oocytes, which likely resulted in the use of alternative energy sources. Signs of reactive oxygen species-induced damage and mitochondrial dysfunction have been observed in maturing follicles, and even in early stages of embryogenesis. However, although recent evidence indicates that also primordial follicles have metabolically active mitochondria, it is still often assumed that these follicles avoid oxidative phosphorylation to prevent oxidative damage in dictyate arrested oocytes. Data on the influence of ovarian aging on oocyte metabolism and mitochondrial function are still limited. A set of 39 formalin-fixed and paraffin-embedded ovarian tissue biopsies were divided into different age groups and used for immunofluorescence analysis of oxidative phosphorylation activity and oxidative damage to proteins, lipids, and DNA. Additionally, 150 immature oocytes (90 germinal vesicle oocytes and 60 metaphase I oocytes) and 15 cumulus cell samples were divided into different age groups and used for targeted metabolomics and lipidomics analysis. Ovarian tissues used for immunofluorescence microscopy were collected through PALGA, the nationwide network, and registry of histo- and cytopathology in The Netherlands. Comprehensive metabolomics and lipidomics were performed by liquid-liquid extraction and full-scan mass spectrometry, using oocytes and cumulus cells of women undergoing ICSI treatment based on male or tubal factor infertility, or fertility preservation for non-medical reasons. Immunofluorescence imaging on human ovarian tissue indicated oxidative damage by protein and lipid (per)oxidation already at the primordial follicle stage. Metabolomics and lipidomics analysis of oocytes and cumulus cells in advanced maternal-age groups demonstrated a shift in the glutathione-to-oxiglutathione ratio and depletion of phospholipids. Age-related changes in polar metabolites suggested a decrease in mitochondrial function, as demonstrated by NAD+, purine, and pyrimidine depletion, while glycolysis substrates and glutamine accumulated, with age. Oocytes from women of advanced maternal age appeared to use alternative energy sources like glycolysis and the adenosine salvage pathway, and possibly ATP which showed increased production in cumulus cells. The immature oocytes used in this study were all subjected to ovarian stimulation with high doses of follicle-stimulating hormones, which might have concealed some age-related differences. Further studies on how to improve mitochondrial function, or lower oxidative damage, in oocytes from women of advanced maternal age, for instance by supplementation of NAD+ precursors to promote mitochondrial biogenesis, are warranted. In addition, supplementing the embryo medium of advanced maternal-age embryos with such compounds could be a treatment option worth exploring. The study was funded by the Amsterdam UMC. The authors declare to have no competing interests. N/A.

Association of Human Papilloma Virus and Epstein-Barr Virus with Ovarian Cancer in Shiraz, Southwestern Iran.

Background & Objective:Ovarian cancer is one of the most common cancers amongst women. The association of Human papillomavirus (HPV) and Epstein-Barr virus (EBV) with ovarian cancer is inconclusive; therefore, the aims of this study were to evaluate the frequency of HPV and EBV in malignant, borderline, benign and normal ovarian tissues.Methods:In this case-control study, 205 Paraffin-embedded ovarian tissue specimens including 68 malignant, 27 borderline, 65 benign, and 45 normal tissues were included from December 2014 to January 2018 and subjected to DNA extraction. The β-globin gene was amplified using PCR to confirm the quality of the extracted DNA. The genomes of HPV (genotypes 16 and 18) and EBV were identified, using specific primers by PCR.Results:The mean age of participants was 43.42 ± 15.4 years. The frequency of HPV was statistically significant between malignant versus benign (P=0.02) and control groups (P=0.002), but not with borderline tumor group (P=0.78). Amongst HPV infected samples, 1 (4.5%) and 14 (63.6%) samples were infected with types 16 and 18, respectively. Also 4 (18.2 %) samples were infected with both genotypes. Eleven samples including 7(10.3%) malignant, 1 (3.7%) borderline, 3 (4.6%) benign and none (0%) of normal control groups were infected with EBV, which was statistically different between malignant and the normal control group (P=0.03).Conclusion:The results of our study showed the possible role of high risk HPVs as well as EBV in pathogenesis of ovarian cancer, and further studies are recommended to confirm these findings.

Histological and immunohistochemical investigations of ovarian interstitial glands during non-breeding season in camels (Camelus dromedarius).

The aim of this was to investigate the histology and immunohistochemistry of interstitial glands during non-breeding season in camel ovaries. A total of 21 mature, non-pregnant and apparently healthy camels aged between 8 and 12years were slaughtered. The ovaries were removed within 15min, cleaned from adipose tissue, weighted and examined grossly. The histological preparation was made, and then, the blocks were cut at 3-5microns thickness and stained by H&E for histological examinations. Moreover, some sections were stained with Sudan Black for lipid detection. Immunohistochemical analysis of paraffin-embedded ovarian tissues was performed to detect the localization of S-100, vimentin, progesterone receptors (PR) and oestrogen receptors (ER). Immunoreactive signals were detected using UltraVision Detection System. The results revealed that the interstitial glands were located in the cortical region and they were arranged in various arrangements either single, in couple or in groups rich in lipid droplet. All interstitial gland arrangements were enclosed by connective tissue capsules containing fibroblasts and collagenous fibres separated them from the surrounding ovarian structures. Both interstitial glands and their surrounding CT were penetrated by several blood vessels. There was a strong immunoreactive signal for S-100 in the nuclei of interstitial cells, and no signals were detected either in cells of the interstitial glands or their connective tissue with PR. We could conclude that the interstitial gland is distinct in ovary of camel and further studies are needed to elucidate its rule in steroid synthesis.

Protective effect of curcumin on ovarian reserve in a rat ischemia model: an experimental study

To determine the protective effect of curcumin on ovarian reserve in a rat ischemia model. Thirty female Albino rats were randomly divided into two groups by time of unilateral, left ovary ischemia/reperfusion (group 1: two-hour ischemia / two-hour reperfusion; group 2: four-hour ischemia / four-hour reperfusion). Each group was subdivided into three subgroups, sham, control, and curcumin (intraperitoneal curcumin (200 mg/kg) simultaneously with reperfusion). Histological grading of ischemic indices of paraffin-embedded ovarian tissue using hematoxylin and eosin (H&E), and anti-Mulerian hormone (AMH) levels by enzyme-linked immunosorbent assay (ELISA), were measured 40 days later. No difference was found between groups 1 and 2 or among subgroups within either group for right and left ovary grades. AMH levels were significantly higher in the curcumin subgroup compared to sham and control within group 2 and in group 2 versus group 1 curcumin subgroups. Curcumin maintains and protects ovarian functions in an ischemia-reperfusion rat model.

Correlation of CD44v6 and type IV collagen immunohistochemical expression in borderline and malignant surface epithelial tumors

Background: the difference in expression of type IV collagen in borderline tumors and ovarian carcinomas has been studied, but the association with adhesion molecules like CD44 have not gain enough interest. Objectives: The purpose of this study is to assess the expression of CD44v6 and type IV collagen status in borderline tumors and invasive ovarian carcinomas and the correlation between them to define the role of these molecules in tumor invasion and metastasis. Type of the study: A cross sectional study Methods: The study included a total of (101) formalin-fixed paraffin-embedded ovarian tissue blocks; of which (19) cases were borderline tumors and (82) cases were overt ovarian carcinomas. Sections from each block were immunohistochemically stained for CD44v6 and type IV collagen. Results: CD44v6 was significantly correlated with FIGO stage of borderline tumors (p=0.001) and ovarian carcinoma (<0.001) and with histological grade of ovarian carcinomas (p=0.004). There was significantly higher expression of type IV collagen in borderline tumors compared to invasive carcinoma(p<0.001) this significance was also seen in correlation to age, stage and grade of the invasive carcinoma, no significant differences in other clinicopathological features were found. There was negative correlation between CD44 v6 & type IV collagen which was statistically significance (P<0.05) in carcinoma but not in borderline tumors. Conclusions: Our data suggest that observed inverse correlation of type IV collagen expression with CD44v6 positivity in surface epithelial tumors indicates that these molecules may cooperate in the invasion and progression of ovarian carcinomas.

Analysis of the gene expression profile in response to human epididymis protein 4 in epithelial ovarian cancer cells.

Currently, there are emerging multiple studies on human epididymis protein4 (HE4) in ovarian cancer. HE4 possesses higher sensitivity and specificity than CA125 in the confirmative early diagnosis for ovarian cancer. Although much attention has been given to explore its clinical application, research of the basic mechanisms of HE4 in ovarian cancer are still unclear. In the present study, we provide fundamental data to identify full-scale differentially expressed genes (DEGs) in response to HE4 by use of human whole-genome microarrays in human epithelial ovarian cancer cell line ES-2 following overexpression and silencing of HE4. We found that a total of 717genes were upregulated and 898genes were downregulated in the HE4-overexpressing cells vs. the HE4-Mock cells, and 166genes were upregulated and 285were downregulated in the HE4-silenced cells vs. the HE4-Mock cells. An overlap of 16genes consistently upregulated and 8genes downregulated in response to HE4 were noted. These DEGs were involved in MAPK, steroid biosynthesis, cell cycle, the p53 hypoxia pathway, and focal adhesion pathways. Interaction network analysis predicted that the genes participated in the regulatory connection. Highly differential expression of the FOXA2, SERPIND1, BDKRD1 and IL1A genes was verified by quantitative real-time PCR in 4cell line samples. Finally, SERPIND1 (HCII) was validated at the protein level by immunohistochemistry in 107paraffin-embedded ovarian tissues. We found that SERPIND1 may act as a potential oncogene in the development of ovarian cancer. The present study displayed the most fundamental and full-scale data to show DEGs in response to HE4. These identified genes may provide a theoretical basis for investigations of the underlying molecular mechanism of HE4 in ovarian cancer.

ATR-FTIR spectroscopy coupled with chemometric analysis discriminates normal, borderline and malignant ovarian tissue: classifying subtypes of human cancer.

Surgical management of ovarian tumours largely depends on their histo-pathological diagnosis. Currently, screening for ovarian malignancy with tumour markers in conjunction with radiological investigations has a low specificity for discriminating benign from malignant tumours. Also, pre-operative biopsy of ovarian masses increases the risk of intra-peritoneal dissemination of malignancy. Intra-operative frozen section, although sufficiently accurate in differentiating tumours according to their histological type, increases operation times. This results in increased surgery-related risks to the patient and additional burden to resource allocation. We set out to determine whether attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, combined with chemometric analysis can be applied to discriminate between normal, borderline and malignant ovarian tumours and classify ovarian carcinoma subtypes according to the unique spectral signatures of their molecular composition. Formalin-fixed, paraffin-embedded ovarian tissue blocks were de-waxed, mounted on Low-E slides and desiccated before being analysed using ATR-FTIR spectroscopy. Chemometric analysis in the form of principal component analysis (PCA), successive projection algorithm (SPA) and genetic algorithm (GA), followed by linear discriminant analysis (LDA) of the obtained spectra revealed clear segregation between benign versus borderline versus malignant tumours as well as segregation between different histological tumour subtypes, when these approaches are used in combination. ATR-FTIR spectroscopy coupled with chemometric analysis has the potential to provide a novel diagnostic approach in the accurate diagnosis of ovarian tumours assisting surgical decision making to avoid under-treatment or over-treatment, with minimal impact to the patient.

The MyD88+ phenotype is an adverse prognostic factor in epithelial ovarian cancer.

The prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. Recent evidence points to the Toll-like receptor-4 (TLR4), and particularly its adaptor protein MyD88, as one potential mediator of this resistance. This study aims to provide further evidence that MyD88 positive cancer cells are clinically significant, stem-like and reproducibly detectable for the purposes of prognostic stratification. Expression of TLR4 and MyD88 was assessed immunohistochemically in 198 paraffin-embedded ovarian tissues and in an embryonal carcinoma model of cancer stemness. In parallel, expression of TLR4 and MyD88 mRNA and regulatory microRNAs (miR-21 and miR-146a) was assessed, as well as in a series of chemosensitive and resistant cancer cells lines. Functional analysis of the pathway was assessed in chemoresistant SKOV-3 ovarian cancer cells. TLR4 and MyD88 expression can be reproducibly assessed via immunohistochemistry using a semi-quantitative scoring system. TLR4 expression was present in all ovarian epithelium (normal and neoplastic), whereas MyD88 was restricted to neoplastic cells, independent of tumour grade and associated with reduced progression-free and overall survival, in an immunohistological specific subset of serous carcinomas, p<0.05. MiR-21 and miR-146a expression was significantly increased in MyD88 negative cancers (p<0.05), indicating their participation in regulation. Significant alterations in MyD88 mRNA expression were observed between chemosensitive and chemoresistant cells and tissue. Knockdown of TLR4 in SKOV-3 ovarian cells recovered chemosensitivity. Knockdown of MyD88 alone did not. MyD88 expression was down-regulated in differentiated embryonal carcinoma (NTera2) cells, supporting the MyD88+ cancer stem cell hypothesis. Our findings demonstrate that expression of MyD88 is associated with significantly reduced patient survival and altered microRNA levels and suggest an intact/functioning TLR4/MyD88 pathway is required for acquisition of the chemoresistant phenotype. Ex vivo manipulation of ovarian cancer stem cell (CSC) differentiation can decrease MyD88 expression, providing a potentially valuable CSC model for ovarian cancer.

MicroRNA expression in ovarian carcinoma and its correlation with clinicopathological features

BackgroundMicroRNA (miRNA) expression is known to be deregulated in ovarian carcinomas. However, limited data is available about the miRNA expression pattern for the benign or borderline ovarian tumors as well as differential miRNA expression pattern associated with histological types, grades or clinical stages in ovarian carcinomas. We defined patterns of microRNA expression in tissues from normal, benign, borderline, and malignant ovarian tumors and explored the relationship between frequently deregulated miRNAs and clinicopathologic findings, response to therapy, survival, and association with Her-2/neu status in ovarian carcinomas.MethodsWe measured the expression of nine miRNAs (miR-181d, miR-30a-3p, miR-30c, miR-30d, miR-30e-3p, miR-368, miR-370, miR-493-5p, miR-532-5p) in 171 formalin-fixed, paraffin-embedded ovarian tissue blocks as well as six normal human ovarian surface epithelial (HOSE) cell lines using Taqman-based real-time PCR assays. Her-2/neu overexpression was assessed in ovarian carcinomas (n = 109 cases) by immunohistochemistry analysis.ResultsExpression of four miRNAs (miR-30c, miR-30d, miR-30e-3p, miR-370) was significantly different between carcinomas and benign ovarian tissues as well as between carcinoma and borderline tissues. An additional three miRNAs (miR-181d, miR-30a-3p, miR-532-5p) were significantly different between borderline and carcinoma tissues. Expression of miR-532-5p was significantly lower in borderline than in benign tissues. Among ovarian carcinomas, expression of four miRNAs (miR-30a-3p, miR-30c, miR-30d, miR-30e-3p) was lowest in mucinous and highest in clear cell samples. Expression of miR-30a-3p was higher in well-differentiated compared to poorly differentiated tumors (P = 0.02), and expression of miR-370 was higher in stage I/II compared to stage III/IV samples (P = 0.03). In multivariate analyses, higher expression of miR-181d, miR-30c, miR-30d, and miR-30e-3p was associated with significantly better disease-free or overall survival. Finally, lower expression of miR-30c, miR-30d, miR-30e-3p and miR-532-5p was significantly associated with overexpression of Her-2/neu.ConclusionsAberrant expression of miRNAs is common in ovarian tumor suggesting involvement of miRNA in ovarian tumorigenesis. They are associated with histology, clinical stage, survival and oncogene expression in ovarian carcinoma.

KRAS mutation analysis in genomic DNA isolated from formalin-fixed paraffin-embedded ovarian tissue: evaluation of a strip-based reverse-hybridisation assay

AimsTo evaluate a reverse-hybridisation assay (strip assay) designed for the sensitive detection of 10 mutations in codons 12 and 13 of the KRAS gene. The strip assay relies on mutant-enriched...

KRAS mutation analysis in genomic DNA isolated from formalin-fixed paraffin-embedded ovarian tissue: evaluation of a strip-based reverse-hybridisation assay

KRAS mutation analysis in genomic DNA isolated from formalin-fixed paraffin-embedded ovarian tissue: evaluation of a strip-based reverse-hybridisation assay

Abstract 259: Functional analysis of the β1 subunit of AMP-activated protein kinase in ovarian cancer

Abstract AMP-activated protein kinase (AMPK) is a heterotrimer composed of a catalytic subunit (α) and two regulatory subunits (β and γ). Each subunit has different isoforms, namely, α1, α2, β1, β2, γ1, γ2 and γ3 encoded by distinct genes, which enable the yielding of 12 possible heterotrimeric complexes. Originally known as a cellular energy-balancing sensor, AMPK was recently found to have strong tumour suppression function. Roles of AMPK have recently been extensively studied in metabolic diseases and human cancers. However, the functional aspects of individual subunits of AMPK are barely investigated. Using quantitative RT-PCR, we found that the AMPK-β1 subunit was over-expressed in ovarian cancer as compared with normal ovary. Over-expression of the AMPK-β1 was further confirmed by immunohistochemical staining (IHC) of the paraffin-embedded ovarian tissues. Clinicopathological data analysis indicates that over-expression of AMPK-β1 correlates with low-grade tumour. Furthermore, from the IHC, the geographical location of the AMPK-β1 in ovarian cancer tissues was also known. In addition to its appearance in the cytoplasm, the AMPK-β1 is also presence in the cell membrane. Since the active form of AMPK (phospho-AMPK) only locates at the cytoplasm, we postulate that AMPK-β1 has addition functions other than just component of the AMPK molecule. To determine the role of AMPK-β1 in ovarian cancer, we have carried out functional analysis of the expression of AMPK-β1 in ovarian cancer. Interestingly, AMPK-β1 was found to inhibit cell growth when forced expressed in ovarian cancer cells. Enforced expression of AMPK-β1 can activate AMPK or down-regulate the level of phospho-AKT in ovarian cancer cells. Additional assays are being studied to further determine the role of AMPK-β1 in ovarian cancer. Understanding the functional role of AMPK-β1 may facilitate the design and development of a new strategy for therapeutic intervention. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 259.

Detection of gonadotropin-releasing hormone receptor and its mRNA in primary human epithelial ovarian cancers.

The hypothalamic neuropeptide gonadotropin-releasing hormone (GnRH) serves a key role in regulating mammalian reproductive function. An extrapituitary role for GnRH in the normal and malignant reproductive tissues has been postulated. The purpose of our study is to demonstrate the presence and levels of GnRH receptor (RGnRH) protein and its mRNA in normal and malignant tissues of ovary. Normal human ovarian tissues (n = 13), as well as epithelial ovarian cancer specimens from stages I-IV (n = 39), were obtained from appropriate patients at operation room. Monoclonal antibodies against RGnRH were used for immunohistochemical evaluation of paraffin-embedded ovarian tissue sections by methods of streptavidin-biotin immunostaining. The molecular size and levels of RGnRH were determined by enhanced chemiluminescence-Western blot assay. The amount of RGnRH mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR). The rate of positive immunostaining in ovarian cancers was 53.8% (21/39). The rate of positive staining in the late stage (stages III and IV) was significantly higher than that in the early stage (stages I and II). A single band of molecular weight of about 60 kDa was detected from protein extracts of ovarian cancer as well as from normal ovary. The mean values of fold increase of signal intensities of 60 kDa detected by Western blots in stages I-IV ovarian cancers were 2.39, 2.42, 2.78, and 3.62, respectively, as compared with normal ovarian tissues. The overall positive rate of Western blot analysis for ovarian cancers was 59% (23/39). The mean values of signal intensity of RT-PCR products of RGnRH mRNA in stages I-IV were 2.24, 2.58, 3.10, and 3.20, respectively. The positive rate of overexpression of RGnRH mRNA in ovarian cancer was 70% (21/30). The differences of mean values of signal intensities of Western blot staining (2.41 versus 2.85) as well as RT-PCR products (2.40 versus 3.11) between the early stage and the late stage of ovarian cancers were statistically nonsignificant. Mechanism of autocrine regulation of tumor growth in human epithelial ovarian cancer can be explained by the coexistence of GnRH, RGnRH, and its mRNA, according to our own and other studies. The level of RGnRH expressed by ovarian cancer might be used for targeting chemotherapeutic agents to those patients who harbor RGnRH-positive tumors.

FISH analysis of BRCA1 copy number in paraffin-embedded ovarian cancer tissue samples

We investigated the BRCA1 gene copy number in unselected ovarian malignancies. Both additional genes (amplification) as well as deletion (loss of heterozygosity, LOH) are often thought to have a role in the initiation or progression of cancer. In addition, if there were little change, deletion studies might help identify BRCA1 mutation carriers. Forty-seven paraffin-embedded ovarian tissue blocks obtained between 1984 and 1997 were used for this study. A sample was “deletion-positive” when BRCA1-deleted cells in the tumor area were significantly different from the benign area. Twenty-five (53%) cases were found to be “deletion-positive”. The average age of onset of “deletion-positive” patients was 50.8 years and of “deletion-negative” 57.8 years ( P < 0.05). There was no statistical difference between groups in the staging, histology, or prognosis. A Kaplan–Meier study did show a trend towards poorer survival for “deletion-positive” patients. FISH permits unique molecular characterization of malignancies at a cellular level. Double amplification of HER-2 and c- myc predicts poor ovarian cancer survival. There appears to be a definite role for BRCA1 deletion in reducing the age of ovarian cancer onset and possibly in overall survival. Further FISH studies of this and other patient sets using additional molecular markers are needed.

VEGF and HIF-1α expression are increased in advanced stages of epithelial ovarian cancer

Objective Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that is elevated in epithelial ovarian cancer. Hypoxia inducible factor-1α (HIF-1α) is a transcription factor that plays a regulatory role in the expression of VEGF. Currently, there is limited information regarding VEGF and HIF-1α expression in epithelial ovarian cancer specimens. The objective of this study was to measure VEGF and HIF-1α expression in epithelial ovarian cancer samples and to compare VEGF and HIF-1α expression between ovarian cancer and normal ovarian tissue. Methods Serial sections of paraffin-embedded ovarian tissues from a study group of 16 control patients and 37 patients diagnosed with epithelial ovarian cancer were analyzed for expression of VEGF and HIF-1α using immunohistochemistry. Results There was a strong correlation between the average expression scores of VEGF and HIF-1α expression ( r 2 = 0.991). There was a significant increase in expression of VEGF in stage III and IV tumors over that in controls ( P < 0.005). There was also a significant increase in HIF-1α expression in stage III and IV tumors over that in controls ( P < 0.005 and P < 0.05, respectively). Conclusion VEGF and HIF-1α are both expressed in epithelial ovarian cancer. VEGF expression correlated with HIF-1α expression, suggesting that HIF-1α may contribute to the overexpression of VEGF observed in epithelial ovarian cancer.

Osteopontin as a potential diagnostic biomarker for ovarian cancer.

Development of new biomarkers for ovarian cancer is needed for early detection and disease monitoring. Analyses involving complementary DNA (cDNA) microarray data can be used to identify up-regulated genes in cancer cells, whose products may then be further validated as potential biomarkers. To describe validation studies of an up-regulated gene known as osteopontin, previously identified using a cDNA microarray system. Experimental and cross-sectional studies were conducted involving ovarian cancer and healthy human ovarian surface epithelial cell lines and cultures, archival paraffin-embedded ovarian tissue collected between June 1992 and June 2001, and fresh tissue and preoperative plasma from 144 patients evaluated for a pelvic mass between June 1992 and June 2001 in gynecologic oncology services at 2 US academic institutions. Plasma samples from 107 women selected from an epidemiologic study of ovarian cancer initiated between May 1992 and March 1997 were used as healthy controls. Relative messenger RNA expression in cancer cells and fresh ovarian tissue, measured by real-time polymerase chain reaction as 2(-DeltaDeltaCT)(a quantitative value representing the amount of osteopontin expression); osteopontin production, localized and scored in ovarian healthy and tumor tissue with immunohistochemical studies; and amount of osteopontin in patient vs control plasma, measured using an enzyme-linked immunoassay. The geometric mean for 2(-DeltaDeltaCT)for osteopontin expression in 5 healthy ovarian epithelial cell cultures was 4.1 compared with 270.4 in 14 ovarian cancer cell lines (P =.03). The geometric mean 2(-DeltaDeltaCT)for osteopontin expression in tissue from 2 healthy ovarian epithelial samples was 9.0 compared with 164.0 in 27 microdissected ovarian tumor tissue samples (P =.06). Immunolocalization of osteopontin showed that tissue samples from 61 patients with invasive ovarian cancer and 29 patients with borderline ovarian tumors expressed higher levels of osteopontin than tissue samples from 6 patients with benign tumors and samples of healthy ovarian epithelium from 3 patients (P =.03). Osteopontin levels in plasma were significantly higher (P<.001) in 51 patients with epithelial ovarian cancer (486.5 ng/mL) compared with those of 107 healthy controls (147.1 ng/mL), 46 patients with benign ovarian disease (254.4 ng/mL), and 47 patients with other gynecologic cancers (260.9 ng/mL). Our findings provide evidence for an association between levels of a biomarker, osteopontin, and ovarian cancer and suggest that future research assessing its clinical usefulness would be worthwhile.