Loss of Secreted Frizzled-Related Protein 4 Correlates with an Aggressive Phenotype and Predicts Poor Outcome in Ovarian Cancer Patients

Background: Activation of the Wnt signaling pathway is implicated in aberrant cellular proliferation in various cancers. In 40% of endometrioid ovarian cancers, constitutive activation of the pathway is due to oncogenic mutations in β-catenin or other inactivating mutations in key negative regulators. Secreted frizzled-related protein 4 (SFRP4) has been proposed to have inhibitory activity through binding and sequestering Wnt ligands. Methodology/Principal Findings: We performed RT-qPCR and Western-blotting in primary cultures and ovarian cell lines for SFRP4 and its key downstream regulators activated β-catenin, β-catenin and GSK3β. SFRP4 was then examined by immunohistochemistry in a cohort of 721 patients and due to its proposed secretory function, in plasma, presenting the first ELISA for SFRP4. SFRP4 was most highly expressed in tubal epithelium and decreased with malignant transformation, both on RNA and on protein level, where it was even more profound in the membrane fraction (p<0.0001). SFRP4 was expressed on the protein level in all histotypes of ovarian cancer but was decreased from borderline tumors to cancers and with loss of cellular differentiation. Loss of membrane expression was an independent predictor of poor survival in ovarian cancer patients (p = 0.02 unadjusted; p = 0.089 adjusted), which increased the risk of a patient to die from this disease by the factor 1.8. Conclusions/Significance: Our results support a role for SFRP4 as a tumor suppressor gene in ovarian cancers via inhibition of the Wnt signaling pathway. This has not only predictive implications but could also facilitate a therapeutic role using epigenetic targets

MAL2 and tumor protein D52 (TPD52) are frequently overexpressed in ovarian carcinoma, but differentially associated with histological subtype and patient outcome

Background: The four-transmembrane MAL2 protein is frequently overexpressed in breast carcinoma, and MAL2 overexpression is associated with gain of the corresponding locus at chromosome 8q24.12. Independent expression microarray studies predict MAL2 overexpression in ovarian carcinoma, but these had remained unconfirmed. MAL2 binds tumor protein D52 (TPD52), which is frequently overexpressed in ovarian carcinoma, but the clinical significance of MAL2 and TPD52 overexpression was unknown. Methods: Immunohistochemical analyses of MAL2 and TPD52 expression were performed using tissue microarray sections including benign, borderline and malignant epithelial ovarian tumours. Inmmunohistochemical staining intensity and distribution was assessed both visually and digitally. Results: MAL2 and TPD52 were significantly overexpressed in high-grade serous carcinomas compared with serous borderline tumours. MAL2 expression was highest in serous carcinomas relative to other histological subtypes, whereas TPD52 expression was highest in clear cell carcinomas. MAL2 expression was not related to patient survival, however high-level TPD52 staining was significantly associated with improved overall survival in patients with stage III serous ovarian carcinoma (log-rank test, p < 0.001; n = 124) and was an independent predictor of survival in the overall carcinoma cohort (hazard ratio (HR), 0.498; 95% confidence interval (CI), 0.34-0.728; p < 0.001; n = 221), and in serous carcinomas (HR, 0.440; 95% CI, 0.294-0.658; p < 0.001; n = 182). Conclusions: MAL2 is frequently overexpressed in ovarian carcinoma, and TPD52 overexpression is a favourable independent prognostic marker of potential value in the management of ovarian carcinoma patients.11 page(s

A clinicopathological review of 33 patients with vulvar melanoma identifies c-KIT as a prognostic marker

Vulvar melanoma is the second most common vulvar cancer. Patients with vulvar melanoma usually present with the disease at a late stage and have a poor prognosis. The prognostic predictors reported in the literature are not unequivocal and the role of lichen sclerosus and c-KIT mutations in the aetiology of vulvar melanoma is unclear. Breslow staging currently seems to be the most adequate predictor of prognosis. We thus performed a clinicopathological and literature review to identify suitable predictors of prognosis and survival and investigated the expression of c-KIT (by immunohistochemistry) in patients with vulvar melanoma (n=33) from the Gynaecological Cancer Centres of the Royal Hospital for Women (Sydney, Australia) and John Hunter Hospital (Newcastle, Australia). Our series of 33 patients fitted the expected clinical profile of older women: delayed presentation, high stage, limited response to treatment and poor prognosis. We identified 3 patients (9.1%) with lichen sclerosus associated with melanoma in situ, although no lichen sclerosus was found in the areas of invasive melanoma. No patient had vulvar nevi. We identified a) Breslow's depth, b) an absence of any of the pathological risk factors, such as satellitosis, in-transit metastasis, lymphovascular space invasion (LVSI) and dermal mitosis, c) removal of inguino-femoral lymph nodes, d) lateral margin of <1 cm, and e) c-KIT expression as valuable prognostic predictors for disease-free survival. We conclude that c-KIT expression is, apart from Breslow's depth, another valuable predictor of prognosis and survival. Lichen sclerosus may be associated with vulvar melanoma

MAL2 and tumor protein D52 (TPD52) are frequently overexpressed in ovarian carcinoma, but differentially associated with histological subtype and patient outcome

Abstract Background The four-transmembrane MAL2 protein is frequently overexpressed in breast carcinoma, and MAL2 overexpression is associated with gain of the corresponding locus at chromosome 8q24.12. Independent expression microarray studies predict MAL2 overexpression in ovarian carcinoma, but these had remained unconfirmed. MAL2 binds tumor protein D52 (TPD52), which is frequently overexpressed in ovarian carcinoma, but the clinical significance of MAL2 and TPD52 overexpression was unknown. Methods Immunohistochemical analyses of MAL2 and TPD52 expression were performed using tissue microarray sections including benign, borderline and malignant epithelial ovarian tumours. Inmmunohistochemical staining intensity and distribution was assessed both visually and digitally. Results MAL2 and TPD52 were significantly overexpressed in high-grade serous carcinomas compared with serous borderline tumours. MAL2 expression was highest in serous carcinomas relative to other histological subtypes, whereas TPD52 expression was highest in clear cell carcinomas. MAL2 expression was not related to patient survival, however high-level TPD52 staining was significantly associated with improved overall survival in patients with stage III serous ovarian carcinoma (log-rank test, p Conclusions MAL2 is frequently overexpressed in ovarian carcinoma, and TPD52 overexpression is a favourable independent prognostic marker of potential value in the management of ovarian carcinoma patients.</p

Low meprin alpha expression differentiates primary ovarian mucinous carcinoma from gastrointestinal cancers that commonly metastasise to the ovaries

Background: Currently, no specific immunohistochemical markers are available to differentiate primary mucinous epithelial ovarian cancer (MOC) from adenocarcinomas originating at other sites that have metastasised to the ovary, which may have an impact on patient management and prognosis. Aim: To investigate the expression of two intestinal markers, galectin 4 and meprin α, in mucinous carcinomas of the ovary and gastrointestinal tract. Methods: Using immunohistochemical analysis, the expression of galectin 4 and meprin α was investigated in 10 MOCs and in 38 mucinous adenocarcinomas of colon, pancreas, stomach and appendix, the most common sites of origin of ovarian metastases. Results: Total cytoplasmic galectin 4 expression was relatively consistent between the different carcinomas. Membranous meprin α expression was significantly lower in MOCs compared with gastrointestinal carcinomas. Moreover, meprin α expression showed greater discrimination between the ovarian and gastrointestinal carcinomas than the cytokeratins CK7 and CK20, the current standard immunohistochemical markers used to determine the tissue origin of mucinous carcinomas involving the ovaries. Conclusions: Meprin α is a useful additional marker in differentiating primary from secondary mucinous adenocarcinomas of the ovary

Prognostic and diagnostic significance of DNA methylation patterns in high grade serous ovarian cancer

Objective: Altered DNA methylation patterns hold promise as cancer biomarkers. In this study we selected a panel of genes which are commonly methylated in a variety of cancers to evaluate their potential application as biomarkers for prognosis and diagnosis in high grade serous ovarian carcinoma (HGSOC); the most common and lethal subtype of ovarian cancer. Methods: The methylation patterns of 10 genes (<i>BRCA1, EN1, DLEC1, HOXA9, RASSF1A, GATA4, GATA5, HSULF1, CDH1, SFN</i>) were examined and compared in a cohort of 80 primary HGSOC and 12 benign ovarian surface epithelium (OSE) samples using methylation-specific headloop suppression PCR. Results: The genes were variably methylated in primary HGSOC, with <i>HOXA9</i> methylation observed in 95% of cases. Most genes were rarely methylated in benign OSE, with the exception of SFN which was methylated in all HGSOC and benign OSE samples examined. Methylation of <i>DLEC1</i> was associated with disease recurrence, independent of tumor stage and suboptimal surgical debulking (HR 3.5 (95% CI:1.10–11.07), p = 0.033). A combination of the methylation status of <i>HOXA9</i> and <i>EN1</i> could discriminate HGSOC from benign OSE with a sensitivity of 98.8% and a specificity of 91.7%, which increased to 100% sensitivity with no loss of specificity when pre-operative CA125 levels were also incorporated. Conclusions: This study provides further evidence to support the feasibility of detecting altered DNA methylation patterns as a potential diagnostic and prognostic approach for HGSOC

SFRP4 expression in cell lines, primary cultures and ascites.

<p>Loss of <i>SFRP4</i> expression from primary tubal epithelial cell lines towards ovarian cancer cell lines is shown in various experimental settings. (A) We have applied fluorescence based RT-qPCR to investigate <i>SFRP4</i> gene expression in primary tubal (healthy patients) and immortalized cell lines. HOSE6-3 was selected as control and expressions of other cell lines were calculated as a ratio compared to HOSE6-3: R = 2^{−[ΔCq<i>SFRP4</i>- ΔCqHOSE6-3]}. (B) SFRP4 and its downstream targets activated β-catenin (ABC), β-catenin and GSK3 β were measured by Western-blot in various cell lines (HOSE6-3, TOV21G (clear cell, Type I ovarian cancer), TOV112D (endometrioid) and SKOV3 (serous) Type II ovarian cancers) as well as in (C) ascites samples from high grade serous ovarian cancer patients with chemoresistance, collected at two consecutive time points during disease progression (TP, time point shown as Western-blot; B2M, beta-2-microglobulin was used as loading control). (D) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031885#s3" target="_blank">Results</a> for SFRP4, activated β-catenin (ABC), β-catenin, GSK3 β are presented quantitatively using densitometric analysis from one experiment on patient ascites samples.</p

SFRP4 protein expression measured by immunohistochemistry.

<p>IHC demonstrating representative SFRP4 protein expression at two magnifications (×10 left column, ×40 right column) in normal tissues (ovarian surface epithelium (A) and tubal epithelium (B)) and various types of ovarian cancers (endometrioid (C), serous (D) and clear cell (E)).</p