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The objective of this study was to analyze the expression and clinical role of the RNA-binding molecule HuR in metastatic high-grade ovarian serous carcinoma (HGSC). HUR mRNA expression by reverse-transcription polymerase chain reaction was analyzed in 66 effusions from patients diagnosed with HGSC. Protein expression was analyzed in 262 HGSC effusions using immunohistochemistry. HUR mRNA was detected in all 66 effusions. HUR mRNA levels were unrelated to clinicopathological parameters. However, higher HUR mRNA levels were significantly related to poor overall survival in the entire cohort (P=.023), as well as in analysis limited to patients with prechemotherapy primary diagnosis specimens (P=.001) in univariate analysis. Cox multivariate analysis showed an independent prognostic role for HUR mRNA in the entire cohort (P=.033) and in patients with prechemotherapy primary diagnosis specimens (P=.002). HuR protein was detected in the nucleus and cytoplasm of tumor cells in 258 (98%) of 262 and 153 (58%) of 262 effusions, respectively. Higher HuR protein expression was associated with higher serum Cancer Antigen (CA) 125 levels at diagnosis (P=.01), but its presence at both cellular compartments was otherwise unrelated to clinicopathological parameters or survival. In conclusion, HuR is widely expressed in metastatic HGSC at both the mRNA and protein level. Higher HUR mRNA levels are associated with poor survival in metastatic HGSC, whereas protein expression has no prognostic value.

We recently identified gene signatures that allow classification of ovarian carcinoma into 5 distinct clinically relevant groups. In the present study, we investigated the clinical role of 10 protein products of the discriminating genes, with focus on epithelial-mesenchymal transition and stem cell markers. Expression of E-cadherin, N-cadherin, P-cadherin, Zeb1, HMGA2, Rab25, CD24, NCAM (CD56), Sox11, and vimentin was assessed in 100 advanced-stage (International Federation of Gynecology and Obstetrics stages III-IV) serous ovarian carcinoma effusions using immunohistochemistry. Results were analyzed for association with clinicopathological parameters, including chemotherapy response, and survival. All 10 proteins were frequently expressed in carcinoma cells. HMGA2 expression was related to older age (P = .015). HMGA2 and NCAM expression was related to stage III disease (P = .011 and P = .023, respectively), and NCAM was overexpressed in peritoneal compared with pleural effusions (P = .001). Vimentin and Zeb1 expression was significantly related to poor chemotherapy response at diagnosis (P = .005 and P = .017, respectively). The associations between NCAM and peritoneal localization and of vimentin and poor chemoresponse were retained after Bonferroni correction. NCAM expression was associated with a trend for shorter overall survival in univariate survival analysis (P = .187), but emerged as an independent prognosticator in Cox multivariate analysis (P = .042). This study identifies vimentin and Zeb1 as markers of poor chemoresponse in metastatic serous ovarian carcinoma effusions and suggests NCAM as potential prognostic marker in metastatic disease. The generally limited prognostic role of the studied markers emphasizes the difficulty in applying data obtained in studies of primary ovarian carcinomas to analyses of ovarian carcinoma effusions, reflecting the unique biology of the latter.

The involvement of VICKZ proteins has been implicated in a large number of cancers. The aim of the present study was to investigate the biological and clinical role of VICKZ proteins in ovarian carcinoma (OC). VICKZ1-3 protein expression was analyzed in 82 serous OC specimens (51 effusions, 14 primary carcinomas, 17 solid metastases) by immunoblotting. Protein localization was studied using immunohistochemistry in 101 tumors (40 effusions, 25 primary carcinomas, 36 solid metastases). The effect of VICKZ silencing using short hairpin RNA on collagenolytic activity and invasion was assessed in ES-2 OC cells. VICKZ2 was the most frequently expressed family member in serous carcinomas. VICKZ levels measured by pan-VICKZ antibody were significantly higher in primary carcinomas and solid metastases compared to effusions (P < .001). In contrast, VICKZ1 and VICKZ2 were overexpressed in effusions compared to primary carcinomas and solid metastases (P = .016 and P = .024, respectively), and higher VICKZ2 expression in effusions was associated with shorter overall survival in univariate analysis (P = .01). All 3 proteins were localized to OC cells by immunohistochemistry, with tumor-specific expression observed for VICKZ1 and VICKZ2. VICKZ silencing in ES-2 cells led to reduced matrix metalloproteinase 9 activity and reduced invasion. In conclusion, VICKZ2 is the most frequently expressed VICKZ family member in serous OCs. VICKZ1 and VICKZ2 are overexpressed in effusions compared to primary carcinomas and solid metastases, suggesting a biological role at this anatomical site, and appear to have a role in proteolysis and invasion. VICKZ2 may be a prognostic marker in ovarian serous carcinoma effusions.

The aim of the present study was to investigate the expression and clinical role of the aurora A and aurora B kinases in primary and metastatic serous ovarian carcinoma. AURKA and AURKB messenger RNA expression was investigated in 178 tumors (88 effusions, 38 primary carcinomas, and 52 solid metastases) from 144 patients with advanced-stage disease using quantitative real-time polymerase chain reaction. Aurora A and aurora B protein expression by immunohistochemistry was additionally analyzed in 147 tumors. Messenger RNA and protein expression at different anatomical sites were studied for association with clinicopathologic parameters, including chemotherapy resistance and survival. AURKA and AURKB messenger RNA and their protein product were demonstrated in all primary carcinomas, solid metastases, and effusions. The expression of AURKA messenger RNA and aurora A protein was higher in effusions compared with solid specimens (P = .003 and P = .006, respectively). AURKB messenger RNA expression was higher in primary carcinomas, and solid metastases obtained prechemotherapy compared with postchemotherapy (P.001 and P = .012, respectively), with no such difference in effusions (P.05). Low aurora B protein expression was associated with primary chemotherapy resistance (P = .006) and poor treatment response (P = .013) in prechemotherapy effusions. No significant association was found between messenger RNA levels or protein expression and progression-free or overall survival. The present study documents for the first time frequent aurora A and aurora B expression in metastatic ovarian carcinoma, suggesting a role in cancer progression, with higher aurora A expression in effusions compared with primary carcinomas and solid metastases. Low AURKB messenger RNA expression in prechemotherapy effusions might be predictive of intrinsic chemotherapy resistance.

Summary The objective of this study was to analyze the clinical role of 9 microRNAs (miRs) previously found to be overexpressed in ovarian carcinoma effusions compared with primary ovarian carcinomas. High-grade serous carcinoma effusions (n=148) were analyzed for expression of miR-29a, miR-31, miR-99b, miR-182, miR-210, miR-221, miR-222, miR-224, and miR-342 using quantitative polymerase chain reaction. Expression levels were analyzed for association with clinicopathological parameters and survival. miR-29a and miR-31 levels were further assessed for association with protein expression of their targets Stathmin and DNA methyltransferase-3A (DNMT3A) by immunohistochemistry and Western blotting, respectively. miRNA levels were unrelated to clinicopathological parameters. However, higher miR-29a levels were significantly related to longer overall survival in univariate ( P =.007) and Cox multivariate survival analysis ( P =.045). miR-29a levels were inversely related to those of its target DNMT3A ( P =.048), and higher DNMT3A expression was significantly related to poor overall survival in univariate ( P =.03) and Cox multivariate ( P =.016) survival analysis. In contrast, miR-31 levels were directly related to cytoplasmic phospho-Stathmin expression ( P =.029) and unrelated to Stathmin and nuclear phospho-Stathmin, and both Stathmin and phospho-Stathmin expressions were unrelated to survival. miR-29a and its target DNMT3A are novel candidate biomarkers of longer and shorter survival, respectively, in metastatic high-grade serous carcinoma.

The AKT signaling pathway is crucial for cancer cell survival. The objective of this study was to analyze the expression and clinical role of this pathway in serous ovarian carcinoma. Phospho-AKT and phospho-mammalian target of rapamycin protein expression was studied in 269 ovarian carcinomas (159 effusions, 38 primary carcinomas, 72 solid metastases) using immunohistochemistry. The association between AKT, mammalian target of rapamycin, and DJ-1 in effusions was quantitatively analyzed using flow cytometry. AKT phosphorylation status in effusions was further studied using Western blotting. Phospho-AKT and phospho-mammalian target of rapamycin were detected in the majority of tumors at all anatomical sites. Phospho-AKT expression in effusions was higher in grade 3 versus grades 1 and 2 tumors (P = .013). Flow cytometry analysis showed association between AKT, mammalian target of rapamycin, and DJ-1 expression (P < .001). Higher phospho-AKT Thr308/pan-AKT ratio by Western blotting was associated with more advanced International Federation of Gynecology and Obstetrics stage (P = .018) and a trend for poor response to chemotherapy at first disease recurrence (P = .051). Higher phospho-mammalian target of rapamycin protein expression in effusions by immunohistochemistry was associated with poor progression-free survival for patients with postchemotherapy effusions (P = .005). Phospho-mammalian target of rapamycin was an independent predictor of poor progression-free survival for patients with postchemotherapy effusions (P = .03). The association between activated AKT and mammalian target of rapamycin expression and clinicopathologic parameters of aggressive disease, including shorter patient survival, provides further evidence regarding the central role of this signaling pathway in ovarian carcinoma.

Leiomyosarcoma (LMS) is the most common uterine sarcoma. Although the disease is relatively rare, it is responsible for considerable mortality due to frequent metastasis and chemoresistance. The molecular events related to LMS metastasis are unknown to date. The present study compared the global gene expression patterns of primary uterine LMSs and LMS metastases. Gene expression profiles of 13 primary and 15 metastatic uterine LMSs were analyzed using the HumanRef-8 BeadChip from Illumina. Differentially expressed candidate genes were validated using quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry. To identify differently expressed genes between primary and metastatic tumors, we performed one-way analysis of variance with Benjamini-Hochberg correction. This led to identification of 203 unique probes that were significantly differentially expressed in the 2 tumor groups by greater than 1.58-fold with P < .01, of which 94 and 109 were overexpressed in primary and metastatic LMSs, respectively. Genes overexpressed in primary uterine LMSs included OSTN, NLGN4X, NLGN1, SLITRK4, MASP1, XRN2, ASS1, RORB, HRASLS, and TSPAN7. Genes overexpressed in LMS metastases included TNNT1, FOLR3, TDO2, CRYM, GJA1, TSPAN10, THBS1, SGK1, SHMT1, EGR2, and AGT. Quantitative real-time PCR confirmed significant anatomical site-related differences in FOLR3, OSTN, and NLGN4X levels; and immunohistochemistry showed significant differences in TDO2 expression. Gene expression profiling differentiates primary uterine LMSs from LMS metastases. The molecular signatures unique to primary and metastatic LMSs may aid in understanding tumor progression in this cancer and in providing a molecular basis for prognostic studies and therapeutic target discovery.

Osteopontin, a soluble protein present in all body fluids, is involved in signaling pathways related to adhesion and extracellular matrix interactions, affecting multiple cellular functions, including inflammation, angiogenesis, and tumor metastasis. We studied osteopontin expression by immunohistochemistry and its clinical relevance in 170 effusions (140 peritoneal, 30 pleural) from women with advanced-stage ovarian carcinoma. Carcinoma cells expressed osteopontin in 126 (74%) of 170 effusions. Osteopontin expression was more frequent in effusions from patients with high-grade tumors (P = .036) but was significantly associated with better debulking at primary surgery (P = .019) and complete response to chemotherapy at diagnosis (P = .021). Osteopontin expression was positively associated to that of the previously studied nuclear factor κB inhibitor IκB (P = .019) and negatively related to expression of the inhibitor of apoptosis family member XIAP (P = .008) and the angiogenic marker endoglin (CD105; P = .018). In univariate survival analysis, the presence of osteopontin in carcinoma cells in primary diagnosis prechemotherapy effusions was associated with longer progression-free survival (P = .037), a finding that did not retain its significance in multivariate Cox analysis. This study demonstrates that osteopontin is frequently expressed in ovarian carcinoma effusions. However, its presence in tumor cells at this anatomical site is unexpectedly associated with less aggressive clinical course, suggesting different and yet undefined biological roles for this protein in serous effusions.

We recently reported on higher expression of the insulin-like growth factor pathway genes IGF-II and IGFBP3 in serous ovarian/peritoneal carcinoma compared to malignant peritoneal mesothelioma. The present study analyzed the diagnostic and clinical role of these proteins in serous effusions. Effusions (n = 327), including 294 carcinomas (205 ovarian, 48 breast, 17 cervical/endometrial, 12 lung, 12 gastrointestinal/genitourinary) and 33 malignant mesotheliomas, were immunostained for insulin-like growth factor-II and insulin-like growth factor binding protein-3. Surgical ovarian carcinoma (n = 124) and peritoneal mesothelioma (n = 18) specimens were additionally studied. Insulin-like growth factor binding protein-3 levels were measured in 148 effusion supernatants (114 ovarian carcinomas, 18 breast carcinomas, 16 mesotheliomas) using enzyme-linked immunosorbent assay. Insulin-like growth factor binding protein-3 promoter methylation was analyzed in 11 ovarian carcinoma effusions. Insulin-like growth factor binding protein-3 (P = .002) and insulin-like growth factor-II (P < .001) expression by immunohistochemistry was significantly higher in carcinomas compared to mesotheliomas, with diagnostic sensitivity of 77% and 70% and specificity of 55% and 70%, respectively. In surgical specimens, insulin-like growth factor binding protein-3 expression was higher in ovarian carcinomas compared to peritoneal mesotheliomas (P = .007), whereas insulin-like growth factor-II expression was comparable (P = .505). Insulin-like growth factor binding protein-3 levels by enzyme-linked immunosorbent assay were comparable in the 3 analyzed cancer types. Insulin-like growth factor binding protein-3 promoter methylation was found in 6 of 11 effusions. High insulin-like growth factor binding protein-3 expression in prechemotherapy and high insulin-like growth factor-II expression in postchemotherapy ovarian carcinoma effusions correlated with poor overall survival (P = .031 and P = .024, respectively). Insulin-like growth factor-II expression in postchemotherapy effusions was an independent prognostic factor in Cox multivariate analysis (P = .04). In conclusion, insulin-like growth factor-II and insulin-like growth factor binding protein-3 are more frequently expressed in metastatic carcinomas compared to mesothelioma in effusions but are less specific than currently used markers. Insulin-like growth factor-II and insulin-like growth factor binding protein-3 may be novel prognostic markers in metastatic ovarian carcinoma.

Tumor progression and treatment failure in ovarian carcinoma are frequently associated with metastasis to effusions. The present study analyzed the expression and clinical role of nuclear factor-kappaB p65, nuclear factor-kappaB inhibitor alpha, and parameters of apoptosis in serous carcinoma. Cleaved caspase-3 and caspase-8 levels and deoxyuridine triphosphate incorporation were measured in 65 effusions using flow cytometry. Effusions (n = 209) and corresponding primary carcinomas and solid metastases (n = 114) were immunohistochemically analyzed for nuclear factor-kappaB p65 and nuclear factor-kappaB inhibitor alpha expression. Effusions (n = 75) were further analyzed for nuclear factor-kappaB phospho-p65 (Ser536) levels using immunoblotting. Results were analyzed for association with anatomic site, clinicopathologic parameters, and survival. Caspase cleavage and deoxyuridine triphosphate incorporation were limited to less than 10% of cells in most effusions. Nuclear factor-kappaB p65 expression was frequently detected at all anatomic sites, with less frequent cytoplasmic nuclear factor-kappaB p65 and nuclear factor-kappaB inhibitor alpha expressions. Immunoblotting showed nuclear factor-kappaB p65 phosphorylation in 72 (96%) of 75 effusions. Higher than median cleaved caspase-3 levels correlated with improved overall and progression-free survival in univariate analysis of all patients (P = .024 and P = .046, respectively) and of those with postchemotherapy effusions (P = .042 and P = .036, respectively). Cleaved caspase-3 expression was an independent predictor of longer progression-free survival for patients with postchemotherapy effusions (P = .029). Nuclear factor-kappaB p65 expression correlated with poor progression-free survival for all patients (P = .048) and for those with postchemotherapy effusions (P = .025). Ovarian carcinoma cells in effusions undergo little apoptosis, but high levels of cleaved caspase-3 are associated with improved survival. Nuclear factor-kappaB p65 is frequently expressed in advanced-stage serous ovarian carcinoma, and its nuclear localization is associated with poor progression-free survival.

Claudins are tight junction proteins that are highly expressed in ovarian carcinoma (OC). The objective of this study was to analyze the anatomic site-related expression and clinical role of claudins in OC. Effusions (n = 218), corresponding primary tumors (n = 81), and solid metastases (n = 164) (total = 463 tumors) were immunostained for claudin-1, claudin-3, claudin-4, and claudin-7. Results were analyzed for association with anatomic site, clinicopathologic parameters, and survival. All 4 claudins were expressed in >85% of tumors at all anatomic sites. However, staining extent of all except claudin-4 was significantly higher in effusions compared with both primary carcinomas and solid metastases (P < .001). In univariate survival analysis of the entire cohort, higher claudin-3 (P = .038) and claudin-7 (P = .035) expression in effusions correlated with shorter overall survival (OS), with similar results for claudin-7 in analysis of progression-free survival (P = .026). In separate analysis for patients with prechemotherapy effusions, higher claudin-7 expression correlated with shorter OS (P = .045). For patients with postchemotherapy effusions, higher claudin-1 (P = .018) and claudin-3 (P = .009) expression correlated with shorter OS. In multivariate survival analysis of the entire cohort, claudin-7 expression was an independent predictor of poor progression-free survival (P = .017). Claudin-3 independently predicted poor OS for patients with postchemotherapy effusions (P = .012). With the exception of claudin-4, claudins are upregulated in OC effusions compared with solid tumors, in agreement with our previous data for cadherins and integrins in this cancer type, suggesting a prosurvival role for these surface molecules. Claudin-3 and claudin-7 expression in effusions independently predicts poor survival in OC.