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The aim of this study was to analyze the expression, biological role and clinical relevance of autotaxin (ATX), the enzyme synthetizing lysophosphatidic acid (LPA), and LPA receptors (LPAR) in high-grade serous carcinoma (HGSC). mRNA expression by qRT-PCR of LPAR1-6 was analyzed in 155 HGSC specimens (88 effusions, 67 solid lesions). ATX mRNA expression was analyzed in 97 specimens. ATX, ERK, and AKT protein expression was studied by Western blotting. LPAR2 mRNA was overexpressed in HGSC cells in effusions compared to solid lesions, with opposite findings for LPAR3 and LPAR6 mRNA and ATX protein. Higher LPAR1 levels were significantly related to longer overall survival (OS) in pre-chemotherapy effusions (p = 0.027). Conversely, higher expression of LPAR1, LPAR2, and LPAR5 in post-chemotherapy effusions was significantly associated with shorter OS (p = 0.037, p = 0.025 and p = 0.021, respectively) and progression-free survival (PFS) (p < 0.001, p = 0.007 and p < 0.001, respectively) in univariate survival analysis. LPAR1 mRNA expression was an independent prognosticator of OS in patients with pre-chemotherapy effusions and PFS in patients with post-chemotherapy effusions (p = 0.013 both). In conclusion, LPAR mRNA and ATX protein levels are anatomic site-dependent in HGSC and the former are informative of disease outcome.

Ovarian cancer, consisting predominantly of ovarian carcinoma, is the eighth most common cancer in women and the most lethal gynecologic malignancy. Efforts focus on identifying biomarkers which may aid in early diagnosis and reduce mortality, as well as on characterizing therapeutic targets with the aim of circumventing chemoresistance and prolonging survival at advanced-stage disease. MicroRNAs (miRNAs) are small, non-coding RNAs that post-transcriptionally regulate gene expression, and have been found to play an important role in ovarian carcinoma. Recent research has identified multiple miRNAs involved in the biology and progression of the disease, and supports a role for miRNAs as potential biomarkers, predictive markers and prognostic factors. Many of the studies published to date nevertheless suffer from critical weaknesses which affect data quality and reproducibility, including the comparison of normal ovaries to tumor tissue without compensation for the highly discrepant target cell fraction in these two specimen types and the inclusion of carcinomas of different histotypes, non-epithelial tumors or tumors of non-specified histology. These shortcomings highlight the critical role of pathologists as part of the team in the setting of such research. This review summarizes current knowledge in this area and discusses the potential clinical relevance of miRNAs in ovarian carcinoma, with focus on studies of clinical specimens in which tissue selection has been deemed adequate., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: MicroRNAs (miRNAs, miRs) are non-coding RNAs which post-transcriptionally regulate mRNA synthesis. Data regarding the expression and clinical relevance of miRNAs and the miRNA-regulating machinery in ovarian carcinoma has been rapidly expanding in recent years. This review presents current knowledge in this area., Methods: PubMed search was undertaken using the terms 'ovarian' and 'microRNA'., Results: A total of 492 papers were identified, of which approximately 100 were publications in English focusing exclusively or partly on clinical ovarian carcinoma specimens. These studies have identified multiple miRNAs with a potential role in the diagnosis, biology and progression of ovarian carcinoma, as well as on predicting chemoresponse and determining prognosis., Conclusions: The presented data support a clinical role for miRNAs in ovarian carcinoma and suggest that miRNA-regulated pathways may be of relevance for novel therapeutics. Novel technologies offer new possibilities for wide-scale miRNA-based classification of OC. Further genomic research focusing on larger series of tumors of similar histological type in combination with experimental approaches is likely to expand our understanding of the role of miRNAs in this cancer., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The present study investigated the expression and clinical role of hyaluronan synthases (HAS1-3) and hyaluronidases (Hyal1-3) in serous ovarian carcinoma. HAS and HYAL mRNA expression was analyzed in 97 tumors (61 effusions, 27 primary carcinomas, 9 solid metastases) using PCR and further studied for association with clinicopathologic parameters, including survival. HAS1 mRNA was overexpressed in effusions compared to primary carcinomas and solid metastases (p < 0.001), and an alternatively spliced HAS1 was expressed only in effusions. HAS2 mRNA was overexpressed in solid metastases and primary carcinomas compared to effusions (p = 0.043), and HAS3 mRNA was overexpressed in primary carcinomas and effusions compared to solid metastases (p = 0.008). HYAL1 mRNA was absent in all specimens, whereas HYAL2 was expressed as two splice variants, of which HYAL2-var2 was overexpressed in solid metastases compared to effusions and primary carcinomas (p < 0.001). HYAL3 mRNA was expressed as wild-type and variant 1-3 form, the latter more highly in primary carcinomas and effusions compared to solid metastases (p = 0.006). HAS1 mRNA was overexpressed in pre- compared to post-chemotherapy effusions (p < 0.001), with opposite finding for HYAL2-var1 and HYAL3-WT (p = 0.016 and p = 0.024, respectively). Higher HYAL2-var1 and HAS1 splice variant mRNA expression in effusions was associated with longer (p = 0.033) and shorter (p = 0.047) overall survival, respectively. These data are the first to document a role for HAS and Hyal members in tumor progression in ovarian carcinoma, as evidenced by their differential expression as function of anatomic site and chemotherapy exposure, with a possible prognostic role for patients with malignant effusions.

The present study analyzed the expression and clinical role of the protein of regenerating liver (PRL) phosphatase family in ovarian carcinoma. PRL1-3 mRNA expression was studied in 184 tumors (100 effusions, 57 primary carcinomas, 27 solid metastases) using RT-PCR. PRL-3 protein expression was analyzed in 157 tumors by Western blotting. PRL-1 mRNA levels were significantly higher in effusions compared to solid tumors (p < 0.001), and both PRL-1 and PRL-2 were overexpressed in pleural compared to peritoneal effusions (p = 0.001). PRL-3 protein expression was significantly higher in primary diagnosis pre-chemotherapy compared to post-chemotherapy disease recurrence effusions (p = 0.003). PRL-1 mRNA expression in effusions correlated with longer overall survival (p = 0.032), and higher levels of both PRL-1 and PRL-2 mRNA correlated with longer overall survival for patients with pre-chemotherapy effusions (p = 0.022 and p = 0.02, respectively). Analysis of the effect of laminin on PRL-3 expression in ovarian carcinoma cells in vitro showed dose-dependent PRL-3 expression in response to exogenous laminin, mediated by Phospholipase D. In contrast to previous studies associating PRL-3 with poor outcome, our data show that PRL-3 expression has no clinical role in ovarian carcinoma, whereas PRL-1 and PRL-2 expression is associated with longer survival, suggesting that PRL phosphatases may be markers of improved outcome in this cancer.

Ovarian cancer is the most lethal gynecologic malignancy. This is attributed to frequent presentation at late stage, when the tumor has metastasized, as well as to development of chemotherapy resistance along tumor progression. Patients with advanced-stage ovarian carcinoma have widespread intraperitoneal metastases, including the formation of malignant serous effusions within the peritoneal cavity. Pleural effusions constitute the most frequent site of distant metastasis (FIGO stage IV disease). Unlike the majority of solid tumors, particularly at the primary site, cancer cells in effusions are not amenable to surgical removal, and failure in their eradication is one of the main causes of treatment failure. Our research in recent years has demonstrated that a large number of cancer-associated molecules are differentially expressed in effusions compared to primary carcinomas and solid metastases. We have additionally observed that expression of several of these molecules differs between primary diagnosis (pre-chemotherapy) and disease recurrence (post-chemotherapy) specimens, and that they are significantly associated with response to chemotherapy and patient survival. These observations are thought to be related to disease progression, as well as to the unique microenvironment of effusions, and may have impact on the selection of targeted therapy in this cancer. This review discusses our recent observations with respect to the biology of ovarian carcinoma cells in effusions, and focuses on the clinical role of tumor-associated molecules at this anatomic site.

Peroxisome proliferator-activated receptors regulate lipid metabolism, affecting inflammation and cancer. The present study analyzed the anatomical site-related expression and prognostic role of peroxisome proliferator-activated receptors in ovarian carcinoma. Fresh-frozen effusions (n = 79), primary carcinomas (n = 44), and solid metastases (n = 16) were studied for peroxisome proliferator-activated receptor-alpha, -beta, and -gamma messenger RNA expression using reverse transcriptase polymerase chain reaction. Peroxisome proliferator-activated receptor-gamma messenger RNA expression was further assessed in 60 tumors (30 effusions, 20 primary carcinomas, 10 metastases) using in situ hybridization. Peroxisome proliferator-activated receptor-gamma protein expression was immunohistochemically analyzed in 160 effusions. All peroxisome proliferator-activated receptors were expressed in most tumors at all anatomical sites using reverse transcriptase polymerase chain reaction, but peroxisome proliferator-activated receptor-alpha (P = .004) and peroxisome proliferator-activated receptor-beta (P = .002) messenger RNA levels were higher in effusions compared with primary carcinomas and solid metastases. In situ hybridization localized peroxisome proliferator-activated receptor-gamma messenger RNA to carcinoma cells in both effusions and solid lesions. Peroxisome proliferator-activated receptor-gamma protein was detected in carcinoma cells in 102 of 160 (64%) effusions. Higher effusion messenger RNA levels of all peroxisome proliferator-activated receptors were associated with less favorable response to chemotherapy at diagnosis (P = .009). In univariate survival analysis, higher messenger RNA expression of all peroxisome proliferator-activated receptors was associated with poor progression-free (P = .045) and overall (P = .014) survival. Higher peroxisome proliferator-activated receptor-gamma protein expression was similarly associated with poor overall survival for the entire cohort (P = .046) and for patients with disease recurrence effusions (P = .009). Peroxisome proliferator-activated receptors were not independent predictors of survival in Cox multivariate analysis. Peroxisome proliferator-activated receptor members are frequently expressed in ovarian carcinoma, with upregulated expression in effusions. Peroxisome proliferator-activated receptor expression in effusions is associated with poor response to chemotherapy at disease recurrence and poor survival, suggesting a role in tumor biology at this unique microenvironment.

Objective: To investigate the activation of mitogen-activated protein kinases (MAPK) in breast carcinoma effusions and to analyze its relationship to anatomic site and clinical parameters., Methods: Activated MAPK (p-ERK, p-JNK, and p-p38) expression was studied in 42 effusions and 51 corresponding solid tumors (23 primary, 28 metastases) using immunohistochemistry (IHC). Hormone receptor and HER2 status, proliferation (Ki-67), and apoptosis (p85-PARP fragment) were assessed. MAPK levels, activity, and activation ratio (phospho/pan-MAPK ratio) were analyzed in 19 effusions using immunoblotting (IB)., Results: Nuclear expression of p-p38 and p-JNK was significantly higher in effusions compared with both primary tumors (P < 0.001 for p-JNK, P = 0.011 for p-p38) and lymph node metastases (P = 0.003 for p-JNK, P = 0.025 for p-p38) but was not accompanied by apoptosis. IB showed pan-ERK and p-ERK in 18/19 effusions, pan-JNK and p-JNK in 18/19 and 17/19 effusions, respectively, and pan-p38 and p-p38 in 19/19 and 17/19 specimens, respectively. In univariate survival analysis of all cases, advanced disease stage (P = 0.041), previous chemotherapy (P = 0.004), and radiation (P = 0.001) and higher Ki-67 scores (P = 0.01) correlated with worse overall survival (OS). In Cox multivariate analysis, stage (P = 0.018), chemotherapy (P = 0.024), radiation (P = 0.017), and ER status (P = 0.002) were independent prognosticators of OS. Quantitative analysis of IB data showed that higher p38 activation ratio correlates with shorter OS (P = 0.01)., Conclusions: This study presents first evidence of in vivo activation of MAPK in breast carcinoma effusions. The elevated JNK and p38 activation in effusions may be a stress-related mechanism providing breast carcinoma cells with survival advantages rather than a drive towards apoptosis. p38 and Ki-67 may be new prognostic markers for patients with breast cancer effusions.

We studied the role of caveolin-1 in tumor progression and prognosis in serous ovarian carcinoma and the association between caveolin-1 and MDR1 expression. The study involved immunohistochemical analysis for caveolin-1 and P-glycoprotein (P-gp) expression in 75 effusions and 90 solid lesions from ovarian and primary peritoneal carcinoma; in situ hybridization for MDR1 messenger RNA (mRNA) expression in 62 effusions and all 90 tumors; and reverse transcription-polymerase chain reaction (RT-PCR) for caveolin-1 mRNA expression in 23 effusions. Immunohistochemical analysis localized caveolin-1 to the cell membrane in 43 effusions and 24 tumors. P-gp membrane expression was detected in 14 effusions and 11 tumors; MDR1 mRNA, in 20 effusions and 30 tumors. Caveolin-1 mRNA was expressed in 19 effusions. Caveolin-1 protein expression showed no association with that of P-gp protein or MDR1 mRNA. The expression of all markers was similar in carcinoma cells in pleural and peritoneal effusions. Caveolin-1 is a novel diagnostic marker for effusions; expression is moderately elevated in tumor cells in effusions, possibly owing to altered signal transduction and metabolism in cancer cells at this site. Expression seems MDR1 independent.

Matrix metalloproteinase-2 (MMP-2) plays a critical role in tumor cell invasion and metastasis. Inhibitors of this enzyme effectively suppress tumor metastasis in experimental animals and are currently being tested in clinical trials. MMP-2 transcriptional regulation is a part of a delicate balance between the expression of various extracellular matrix (ECM) constituents and ECM degrading enzymes. Halofuginone, a low-molecular-weight quinazolinone alkaloid, is a potent inhibitor of collagen type alpha1 (I) gene expression and ECM deposition. We now report that expression of the MMP-2 gene by murine (MBT2-t50) and human (5637) bladder carcinoma cells is highly susceptible to inhibition by halofuginone. Fifty percent inhibition was obtained in the presence of as little as 50 ng/ml halofuginone. This inhibition is due to an effect of halofuginone on the activity of the MMP-2 promoter, as indicated by a pronounced suppression of chloramphenicol acetyltransferase activity driven by the MMP-2 promoter in transfected MBT2 cells. There was no effect on chloramphenicol acetyltransferase activity driven by SV40 promoter in these cells. Halofuginone-treated cells failed to invade through reconstituted basement-membrane (Matrigel) coated filters, in accordance with the inhibition of MMP-2 gene expression. A marked reduction (80-90%) in the lung colonization of MBT2 bladder carcinoma cells was obtained after the i.v. inoculation of halofuginone-treated cells as compared with the high metastatic activity exhibited by control untreated cells. Under the same conditions, there was almost no effect of halofuginone on the rate of MBT2 cell proliferation. These results indicate that the potent antimetastatic activity of halofuginone is due primarily to a transcriptional suppression of the MMP-2 gene, which results in a decreased enzymatic activity, matrix degradation, and tumor cell extravasation. This is the first description, to our knowledge, of a drug that inhibits experimental metastasis through the inhibition of MMP-2 at the transcriptional level. Combined with its known inhibitory effect on collagen synthesis and ECM deposition, halofuginone is expected to exert a profound anticancerous effect by inhibiting both the primary tumor stromal support and metastatic spread.

Using the Matrigel invasion assay, we have examined the role of cell adhesion and migration in the invasiveness of two cell lines, DM and HSR, derived from the Y1 mouse adrenocortical tumor. The DM cells were metastatic and more invasive (10-fold) than the nonmetastatic HSR cells. The difference in invasiveness could not be ascribed to different levels of secreted type IV collagenolytic activity since HSR cells secreted higher levels of activity. Cells from the metastatic DM line showed greater motility to both laminin and fibronectin when compared to the HSR line. Furthermore, both Matrigel and laminin promoted the attachment and spreading of DM cells but they had little effect on the adhesion of the HSR cells. Electron microscopic examination revealed an increased ruffling of the cell membrane in the metastatic DM line. These studies suggest a role for cell adhesion and migration on the invasion of Matrigel by malignant tumor cells.

Metastatic thyroid carcinoma rarely involves the orbit. A 35-year-old woman with a history of thyroid lobectomy for a "benign" goiter 6 years earlier presented with a right superotemporal mass causing bony erosion of the lateral orbital wall shown by clinical examination and computed tomographic (CT) scanning. Orbital exploration with biopsy showed metastatic thyroid carcinoma. This represents the youngest patient reported with this rare condition.