Overexpression of dedicator of cytokinesis I (Dock180) in ovarian cancer correlated with aggressive phenotype and poor patient survival.

AIMS: Dedicator of cytokinesis I (Dock180) is a novel guanine nucleotide exchange factor for Rho guanosine triphosphates (GTPases) important for cell migration. The aim of this study was to evaluate the role of Dock180 in ovarian carcinogenesis. METHODS AND RESULTS: Using immunohistochemistry, real-time polymerase chain reaction and Western blotting, overexpression of Dock180 RNA and protein was demonstrated in the nucleus and cytoplasm of ovarian cancer cell lines (n = 5) and clinical samples of ovarian borderline tumours (n = 21) and invasive cancers (n = 108) when compared with ovarian epithelial cell lines (n = 3) and benign cystadenomas (n = 10) (P < 0.05). High Dock180 cytoplasmic expression in ovarian cancer (n = 108) was associated significantly with serous histological type, high-grade cancer and advanced stage (P < 0.05), as well as poor overall and disease-free survival (P = 0.004). Using multivariate progression analysis, high Dock180 cytoplasmic expression and advanced cancer stage were found to be independent prognostic factors for short overall survival and disease-free survival (P < 0.05). Exogenous expression of Dock180 by transient transfection enhanced cancer cell migration and invasion, whereas knockdown of Dock180 by an siRNA approach retarded cancer cell migration and invasion in association with down-regulation of matrix metalloproteinase 2. CONCLUSIONS: Our findings suggest that Dock180 contributes to ovarian carcinogenesis and dissemination and is a potential prognostic marker and therapeutic target.

Mitochondrial manganese-superoxide dismutase expression in ovarian cancer: role in cell proliferation and response to oxidative stress.

Superoxide dismutases (SODs) are important antioxidant enzymes responsible for the elimination of superoxide radical (O(2)(-)). The manganese-containing SOD (Mn-SOD) has been suggested to have tumor suppressor function and is located in the mitochondria where the majority of O(2)(-) is generated during respiration. Although increased reactive oxygen species (ROS) in cancer cells has long been recognized, the expression of Mn-SOD in cancer and its role in cancer development remain elusive. The present study used a human tissue microarray to analyze Mn-SOD expression in primary ovarian cancer tissues, benign ovarian lesions, and normal ovary epithelium. Significantly higher levels of Mn-SOD protein expression were detected in the malignant tissues compared with normal tissues (p < 0.05). In experimental systems, suppression of Mn-SOD expression by small interfering RNA caused a 70% increase of superoxide in ovarian cancer cells, leading to stimulation of cell proliferation in vitro and more aggressive tumor growth in vivo. Furthermore, stimulation of mitochondrial O(2)(-) production induced an increase of Mn-SOD expression. Our findings suggest that the increase in Mn-SOD expression in ovarian cancer is a cellular response to intrinsic ROS stress and that scavenging of superoxide by SOD may alleviate the ROS stress and thus reduce the simulating effect of ROS on cell growth.

In situ human telomerase reverse transcriptase expression pattern in normal and neoplastic ovarian tissues.

Telomerase is a ribonuclear protein reverse transcriptase that maintains telomere length in eukaryotic cells. Activation of telomerase has been implicated in human cellular immortalization and carcinogenesis. Telomerase activity in ovarian neoplasm has been studied using polymerase chain reaction (PCR)-based methods and shown to be correlated with malignancy. However, we believe those results must be interpreted with caution because such studies used a heterogeneous mix of cells, including normal cell type known to express telomerase when activated. The present study used in situ hybridization that allows determination of the type of cells expressing telomerase, as well as the intensity of that expression, in ovarian neoplasms. A total of 75 specimens were studied. Epithelial telomerase reverse transcriptase mRNA expression was detected in 28 of 31 epithelial ovarian carcinomas, 1 of 1 malignant granulosa cell tumor, 7 of 9 serous borderline ovarian tumors, 11 of 11 mucinous borderline ovarian tumors, 4 of 5 serous cystadenofibromas, 2 of 4 serous cystadenomas, 8 of 8 mucinous cystadenomas, and 0 of 6 normal ovaries except the corpus luteum. Telomerase expression is heterogeneously found in both benign and malignant epithelial tissues. We conclude that human telomerase reverse transcriptase mRNA expression does not seem to be a reliable marker for clinical use in differentiating between benign and malignant tumors.

Expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) in primary human ovarian carcinoma.

The mitogen-activated protein kinase phosphatase-1, MKP-1 (CL100) is involved in inactivation of MAP-kinase pathways, regulation of stress-responses and suppression of apoptosis. We investigated expression of MKP-1 in 90 cases of primary ovarian tumors, 11 normal ovaries as well as 4 ovarian carcinoma cell lines. Immunohistochemical expression of MKP-1 protein was reduced in tissue from LMP tumors and invasive ovarian carcinomas compared to normal ovaries and cystadenomas. A moderate to strong expression of MKP-1 was detected in 57.6% of invasive ovarian carcinomas. In a descriptive univariate survival analysis, MKP-1 expression was a prognostic marker for shorter progression-free survival of patients with invasive ovarian carcinomas. Patients with carcinomas positive for MKP-1 had a median progression-free survival of only 18.3 months compared to 40.6 months for patients with carcinomas negative for MKP-1 (log-rank test, p = 0.019). Other prognostic parameters for progression-free survival were FIGO stage, grade and pT stage. In an exploratory multivariate analysis, we found that MKP-1 expression as well as FIGO stage and grade were independent prognostic factors for progression-free survival. In contrast to progression-free survival, we did not find any influence of MKP-1 expression on patient overall survival. We investigated expression and regulation of MKP-1 mRNA by Northern Blot in vitro using 4 ovarian carcinoma cell lines (SKOV-3, OVCAR-3, CAOV-3, OAW-42). MKP-1 mRNA was inducible by interleukin-1beta and tumor necrosis factor-alpha in SKOV-3 and OVCAR-3 cells, whereas CAOV-3 and OAW-42 expressed MKP-1 mRNA constitutively. In OVCAR-3 cells MKP-1 mRNA levels were strongly inducible upon treatment of cells with cisplatin. Our data indicate that MKP-1 is expressed in a subset of ovarian carcinomas and regulated by inflammatory mediators. Expression of MKP-1 may be associated with shorter progression-free survival times. Further studies are needed to determine whether MKP-1 expression is a clinically useful marker to estimate patient prognosis as well as the response to chemotherapy.

Frequent activation of AKT2 kinase in human pancreatic carcinomas.

Activation of AKT/protein kinase B promotes a variety of biological activities important in tumorigenesis, such as cell survival and cell cycle progression. We previously demonstrated amplification and overexpression of the AKT2 gene in a subset of human pancreatic carcinomas. In this investigation, we assessed AKT2 catalytic activity in 50 frozen pancreatic tissues (37 carcinomas, four benign tumors and nine normal pancreata) by in vitro kinase assay. Twelve of 37 (32%) pancreatic carcinomas showed markedly elevated levels of AKT2 activity compared to normal pancreata and begin pancreatic tumors. To delineate mechanisms contributing to AKT2 activation in malignant pancreatic tumors, we examined the status of upstream components of the phosphatilydlinositol 3-kinase (PI3K)/AKT pathway. Western blot analysis revealed loss of PTEN protein expression in two of the 12 pancreatic carcinomas with activated AKT2. In vitro PI3K assays demonstrated high levels of PI3K activity in seven carcinoma specimens that showed AKT2 activation. Immunohistochemical staining confirmed high levels of phosphorylated (active) AKT in malignant pancreatic tumors compared to normal pancreata. Overall, these data suggest that upstream perturbations of the PI3K/AKT pathway contribute to frequent activation of AKT2 in pancreatic cancer, which may contribute to the pathogenesis of this highly aggressive form of human malignancy.

Expression of cyclooxygenase-2 and inducible nitric oxide synthase in human ovarian tumors and tumor-associated macrophages.

This study investigates whether and to what extent cyclooxygenase type-2 (COX-2) and inducible nitric oxide-synthase (iNOS), both known to have an immunosuppressive effect, are expressed in human ovarian tumors. Because COX-2 and iNOS can be expressed by activated macrophages, the presence of tumor-associated macrophages and the expression of COX-2 and iNOS by these tumor-associated macrophages were determined. The results obtained may provide insight into the function of COX-2 and iNOS expression by tumors. The expression of COX-2 and iNOS in tumor cells and macrophages was assessed in 18 malignant, 15 borderline, and 14 benign human ovarian tumors by immunohistochemical staining of frozen tissue sections. The intra- and peritumoral macrophages were stained using an anti-CD68 monoclonal antibody. Most of the malignant tumors (15 of 18), 10 of 15 borderline, and 9 of 14 benign tumors showed COX-2 expression in the epithelial cells, a result which indicates that COX-2 expression is not exclusive to malignancy. In addition, COX-2 staining was more intense in the epithelial cells of benign and borderline tumors than in malignant tumors. Weak iNOS staining was observed in 5 of 18 malignant, 4 of 15 borderline, and 5 of 14 benign tumors. The number of tumor-associated macrophages varied widely between the different tumors. The highest number of tumor-associated macrophages (> or =20/0.125 mm(2)) was observed in malignant tumors, whereas low to moderate intra- and peritumoral macrophage infiltration (5-20/0.125 mm(2)) was observed in the borderline and benign tumors. COX-2-positive tumor-associated macrophages were found in 3 of 18 malignant tumors, 7 of 15 borderline tumors, and 1 of 14 benign tumors. The number of COX-2-positive tumor-associated macrophages ranged from 3 to 30% of the total macrophage population. Some malignant (4 of 18), borderline (5 of 15), and benign (2 of 14) tumors contained iNOS-positive macrophages. Notable was that COX-2- and iNOS-positive macrophages were predominantly located in the tumor stroma, the regions between tumor and stroma, and in the lumina of the tumor when located in the tumor tissue. These data indicate that not only malignant but also borderline and benign ovarian tumors can exhibit increased levels of COX-2 and iNOS expression. In addition, a small proportion of the tumor-associated macrophages found in malignant, borderline, and benign tumors seems to be in an activated state, judged by their iNOS and COX-2 expression. This subpopulation of tumor-associated macrophages was invariably located in the tumor stroma or in the lumina of the tumor, specifically suggesting that macrophages outside the tumor can be tumor cytotoxic.

alpha1,6fucosyltransferase is highly and specifically expressed in human ovarian serous adenocarcinomas.

An elevated level of alpha1,6fucosylation in N-glycans represents one of the cancer-related alterations of oligosaccharides and is associated with the metastatic potential of hepatoma cells. However, expression of alpha1,6fucosyltransferase (alpha1,6FucT), which is involved in this aberrant glycosylation, has not been intensively explored in other malignant tumors. We report on a study of the expression of alpha1,6FucT in various types of epithelial ovarian carcinoma tissue, as well as normal ovary, benign and borderline ovarian tumors. The activity assay showed that alpha1,6FucT is highly and specifically elevated in serous adenocarcinomas but not in normal and other ovarian tumor tissues. This elevation was due to enhancement of mRNA expression, as evidenced by Northern blot analysis. Furthermore, we have shown immunohistochemically that alpha1,6FucT expression is localized predominantly in cancer cells. Lectin blot analysis using Lens culinaris agglutinin, which preferentially recognizes alpha1,6fucose residue, suggested that several glycoproteins were likely targets for modification by alpha1, 6fucosylation in serous adenocarcinoma tissues. These findings suggest that the elevated expression of alpha1,6FucT and the resulting modification of N-glycans are distinctive features of this type of ovarian cancer and may be related to the progression of this malignancy.

Differential expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 protein and mRNA in epithelial ovarian tumors.

OBJECTIVE: Matrix metalloproteinase-9 (MMP-9) can degrade gelatin and type IV collagen and is known to play an important role in tumor cell invasion across the basement membrane. The tissue inhibitor of metalloproteinase-1 (TIMP-1) is able to prevent activation of pro-MMP-9 and forms a 1:1 complex with the active form of MMP-9. The aim of the present study was to investigate the expression of MMP-9 and TIMP-1 in benign, borderline, and invasive epithelial ovarian tumors. MATERIALS AND METHODS: A total of 90 patients with epithelial ovarian tumor were treated at the Brigham and Women's Hospital and were used as the study population. Immunohistochemistry and in situ hybridization were performed to detect protein and mRNA expression of MMP-9 and TIMP-1. RESULTS: In the 90 epithelial ovarian tumors tested, MMP-9 expression in tumor cells was found to be significantly enhanced in serous and mucinous ovarian carcinomas compared with benign and borderline tumors. We also observed the immunostaining of MMP-9 in stromal cells of benign, borderline, and invasive epithelial ovarian tumors. Moreover, the expression levels of TIMP-1 in tumor cells were significantly higher in borderline and invasive ovarian tumors than in benign tumors. CONCLUSION: Using an in situ hybridization technique, we disclosed a direct correlation between the presence of mRNA and protein expression for both MMP-9 and TIMP-1. The present data suggest that high levels of MMP-9 protein in invasive epithelial ovarian carcinoma are strongly associated with tumor cell invasion. Enhanced expression of TIMP-1 protein in borderline and invasive tumors indicates that endogenous TIMP-1 protein may play a paradoxical role in ovarian tumor progression.

Alterations in the expression of the DNA repair/redox enzyme APE/ref-1 in epithelial ovarian cancers.

The DNA base excision repair pathway is responsible for the repair of alkylation and oxidative DNA damage. A crucial step in the base excision repair pathway involves the cleavage of an apurinic/apyrimidinic (AP) site in DNA by an AP endonuclease (APE). The major AP endonuclease in mammalian cells is APE/ref-1, a multifunctional enzyme that acts not only as an AP endonuclease but as a redox-modifying factor for a variety of transcription factors. The purpose of this study was to determine the expression of APE/redox factor-1 (ref-1) in ovarian tissues, particularly ovarian cancers. Formalin-fixed, paraffin-embedded specimens of ovarian tissues (normal, various benign conditions, and epithelial cancers) were studied using both polyclonal and monoclonal antibodies to APE/ref-1. The relationship between APE/ref-1 protein levels and DNA repair activity was studied in ovarian Hey and Hey-C2 cell lines using Western blot and a specific AP-site oligonucleotide cleavage assay. Hey and Hey-C2 cells were fractionated, and the nuclear and cytoplasmic extracts were quantitated for protein levels and assessed for APE/ref-1 with Western blot. Normal ovarian tissues consistently demonstrated strong nuclear staining of the surface epithelium, epithelial inclusions, corpora lutea and albicantia, and stroma. Cytoplasmic staining was absent. A similar pattern was seen for benign conditions including endometriosis. Low malignant potential ovarian cancers stained in a pattern similar to normal ovarian and nonneoplastic tissues; however, two specimens also had areas of cytoplasmic staining. Epithelial ovarian cancers were remarkably different from all other ovarian tissues studied. Both nuclear and cytoplasmic staining of the malignant epithelium were seen and ranged from strong to weak, often with considerable staining heterogeneity within the same tumor. The AP-site oligonucleotide cleavage assay indicated that APE/ref-1 protein levels correlate well with DNA repair activity. The increased levels of APE/ref-1 in the Hey-C2 cells was mainly attributable to increased cytoplasmic enzyme. APE/ref-1 immunoreactivity is altered in malignant ovarian tumors. Further studies will determine whether the altered expression and subcellular location reflect changes in redox regulatory functions.

Analysis of telomerase activity in ovarian cystadenomas, low-malignant-potential tumors, and invasive carcinomas.

OBJECTIVE: Inappropriate telomerase expression has been reported to be associated with the development and/or progression of malignancies. Therefore, the purpose of the study was to determine and evaluate the levels of telomerase activity in normal ovary, cystadenomas, low-malignant-potential tumors, and carcinomas of the ovary. METHODS: In the present study, telomerase activity was examined in frozen tissue specimens of normal ovary (n = 6), ovarian cystadenomas (n = 13), ovarian low-malignant-potential (LMP) tumors (n = 12), and ovarian invasive carcinomas (n = 81). Clinicopathological information including age at diagnosis, histological grade, FIGO stage, presence of distant metastasis at diagnosis, and residual disease was available for all patients with ovarian carcinomas (n = 81). Telomerase activity was assessed by the telomeric repeat amplification protocol (TRAP). Arbitrary values were assigned to processivity of telomerase activities based on the molecular weights of the telomeric repeat DNA ladders, and were graded as "negative," "moderate" (< or =99 bp), or "high" (>100 bp) activities. The specificity of telomerase activity was determined by the pretreatment of telomerase-positive control or tumor samples with RNase that led to the abolition of the activity. In addition, to determine the possibility of false negativity due to the presence of telomerase inhibitors, TRAP assay was performed on each of the telomerase-negative specimens by mixing them individually with the telomerase-positive control. RESULTS: Telomerase activity in the progression of ovarian carcinogenesis was evaluated. In comparison with normal ovary/cystadenoma (32%), a much higher frequency of the moderate activity was observed in LMP tumors (67%) or invasive carcinomas (57%), suggesting a close association between the latter two categories. The results reflect a subpopulation of telomerase-positive LMP tumor cells with the potential to develop invasive carcinomas. None of the specimens of the benign or LMP tumors exhibited high activity. In contrast, 18% of ovarian invasive carcinomas showed high telomerase activity (P = 0.013, Fisher exact test) and further 57%, moderate activity (75% in all). A statistically significant difference was observed in the expression of telomerase activity between normal ovary/benign cystadenomas and ovarian invasive carcinomas (P = 0.001, chi(2) test). CONCLUSIONS: The study showed a high prevalence of telomerase activity in LMP tumors or invasive carcinomas, the high levels of telomerase activity being associated exclusively with the invasive ovarian carcinomas. Therefore, the levels of processivity of telomerase activity and evidence of its statistically significant association with ovarian carcinoma suggest its role in the progression of ovarian carcinogenesis.

Differential telomerase activity, expression of the telomerase catalytic sub-unit and telomerase-RNA in ovarian tumors.

Telomerase activity has been found in a variety of malignant tumors but only rarely in benign tumors or normal tissues. In this study, we investigated telomerase activation in 37 ovarian tumors, including benign, borderline and malignant neoplasms. Telomerase activity was detected using the telomeric repeat amplification protocol (TRAP) in 13/16 ovarian carcinomas, 9/10 borderline tumors and 3/11 cystadenomas/fibromas. mRNA expression of the putative human telomerase catalytic sub-unit gene (hTERT) was detected by RT-PCR in 14/15 ovarian carcinomas, 8/10 borderline tumors and 4/11 cystadenomas/fibromas. In situ hybridization was performed to evaluate telomerase-RNA (hTR) expression in the corresponding paraffin-embedded tumors. Variable expression levels of hTR were found over neoplastic tumor cells. The highest levels of hTR expression were found predominantly in ovarian carcinomas. Although the amount of telomerase activity varied, significantly high levels of telomerase activity were found predominantly in ovarian carcinomas. hTERT mRNA expression was closely associated with telomerase activity. These findings suggest that up-regulation of hTERT and hTR is important for telomerase activation during malignant-tumor progression. Telomerase activation might therefore be a valuable diagnostic parameter that could help to identify potentially progressive lesions. However, the diagnostic and therapeutic implications of telomerase activation need to be clarified in clinical trials. Int. J. Cancer (Pred. Oncol.) 84:426-431, 1999.

Overexpression of p34cdc2 protein kinase in epithelial ovarian carcinoma.

OBJECTIVE: To investigate the role of expression of p34cdc2 protein kinase in normal, benign, and malignant ovarian epithelium. MATERIAL AND METHODS: Tissue sections from 24 patients with epithelial ovarian carcinoma (EOC) along with 6 normal ovarian specimens and 12 benign cystadenomas were incubated with mouse IgG monoclonal antibody to human p34cdc2 protein kinase, followed by detection with use of a standard peroxidase-labeled streptavidin-biotin technique. Immunohistochemical staining was graded and compared. Clinical data were also reviewed. RESULTS: Normal surface epithelium and 10 of 12 benign cystadenomas failed to stain for p34cdc2 protein kinase. Of the 24 EOC specimens, however, 19 (79%) stained positively. The staining pattern or intensity was not associated with the histologic grade or surgical stage. CONCLUSION: Expression of p34cdc2 protein kinase is strongly up-regulated in most cases of EOC but not in normal epithelial ovarian tissue or in most cases of benign epithelial tumors evaluated. Therefore, it may be associated with early events in carcinogenesis. Redundant overexpression of cyclin-dependent kinases such as p34cdc2 may contribute to deranged cell cycle progression and proliferation of EOC. Observation of overexpression of p34cdc2 protein kinase in other malignant lesions suggests a common mechanism.

Telomerase activity in benign and malignant epithelial ovarian tumors.

BACKGROUND: Ovarian epithelial tumors include benign lesions lacking invasive and metastatic abilities (cystadenomas) in addition to malignant lesions (carcinomas). An intermediate category, called tumors of low malignant potential (LMP), is also recognized. The merit of this classification is being challenged because the clinical behavior of LMP tumors appears closer to that of cystadenomas than to that of carcinomas. PURPOSE: To verify our hypothesis that the expression of the enzyme telomerase distinguishes these two categories of ovarian epithelial tumors, we examined and compared such expression in ovarian cystadenomas and carcinomas. By examining the expression of telomerase in LMP tumors, we then sought to determine if these tumors were more closely related to cystadenomas or to carcinomas with regard to telomerase expression. METHODS: We examined a total of 64 consecutive ovarian tumors subdivided into 20 carcinomas, 17 LMP tumors, and 27 cystadenomas. We subsequently discarded three of the 27 cystadenomas because of the presence of admixed normal ovarian stroma in those specimens. Tumor subtyping was done without knowledge of the telomerase results, and telomerase assays were likewise interpreted without knowledge of tumor types. Telomerase activity was determined by use of the TRAP (i.e., telomeric repeat amplification protocol) assay. Differences between the proportions of tumors expressing this enzyme in each subgroup were evaluated by use of Fisher's exact test (two-sided). RESULTS: Telomerase activity was detected in all 20 carcinomas and in all 17 LMP tumors examined. In contrast, it was not detected in 19 of the 24 cystadenomas. These differences between rates of telomerase expression in either carcinomas or LMP tumors and those in cystadenomas were statistically significant (P<.0001). All five of the telomerase-positive cystadenomas belonged to a variant called papillary cystadenomas, whereas none of the telomerase-negative cystadenomas belonged to this variant (P<.0001). CONCLUSIONS AND IMPLICATIONS: The presence of telomerase expression in ovarian LMP tumors supports the merit of continuing to separate these tumors from cystadenomas, in spite of their apparent benign clinical course. The finding of telomerase expression in papillary cystadenomas suggests that such tumors may be mechanistically related to LMP tumors and should perhaps be reclassified as variants of LMP tumors. Lack of telomerase expression in ovarian cystadenomas raises questions about the alleged immortality of these tumors because expression of this enzyme is thought to be essential for continuous growth in adult tumors.

Establishment of long-term in vitro cultures of human ovarian cystadenomas and LMP tumors and examination of their spectrum of expression of matrix-degrading proteinases.

OBJECTIVES: To obtain long-term cultures of ovarian cystadenomas and ovarian tumors of low malignant potential (LMP) displaying gene expression patterns similar to those found in vivo and test the hypothesis that such cultures would express different levels of matrix-degrading proteinases than cultured ovarian carcinomas. METHODS: Transfection with an adenoviral expression vector for simian virus 40 (SV40) large T antigen was used to establish long-term cultures of the above tumors. Levels of expression of various genes were evaluated using molecular biological and immunohistochemical approaches. Zymography and reverse zymography were used to examine the activity of various metalloproteinases and plasminogen activators (PA). Two-sided P values for differences in plasminogen activator expression between different cell types were evaluated by Fisher's exact test. RESULTS: Long-term cultures derived from cystadenomas and LMP tumors were obtained which formed colonies on semisolid supports, but were not tumorigenic in nude mice. The cultured cells expressed keratin, estrogen receptor, gonadotropin receptors, BRCA1, and originated from monoclonal populations. There was no apparent association between the malignant phenotype and the expression of either matrix metalloproteinases or tissue inhibitors of metalloproteinases. However, a correlation was seen between this phenotype and expression of urokinase (uPA) and tissue type (tPA) plasminogen activators (P = 0.08 and 0.02 respectively). CONCLUSIONS: The above cell strains provide a useful model for investigating various aspects of the biology of benign ovarian tumors, including their response to steroid and gonadotropin hormones, and the role of specific proteinases in the acquisition of invasive and metastatic abilities.

17 beta-hydroxysteroid dehydrogenase in common epithelial ovarian tumors.

Type 1 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) is an estrogen-metabolizing enzyme that catalyzes the conversion of estrone to the more biologically potent estradiol. We examined the immunolocalization of 17 beta-HSD in five specimens of normal human ovary and in 51 specimens of common epithelial tumors of the ovary to study the expression of 17 beta-HSD in these ovarian neoplasms. 17 beta-HSD immunoreactivity was detected in granulosa cells of dominant preantral follicles but not in the surface epithelium of the normal cycling human ovary. In 12 patients, the enzyme was not expressed in tumor cells of benign cystadenoma. 17 beta-HSD was immunolocalized in the cytoplasm of four specimens (50%) of eight of tumors of low malignant potential and in 20 specimens (67%) of 30 of invasive carcinoma. 17 beta-HSD immunoreactivity was also observed in nonluteinized stromal cells of mucinous tumors (two adenomas of six, three tumors of five with low malignant potential, and two of 10 invasive carcinomas) but not in other histologic types. Type 1 17 beta-HSD expression, therefore, correlated with malignant transformation of the surface epithelium of the human ovary, supporting a possible role in its progression by increasing in situ estradiol concentration.

Tissue and serum metalloproteinase (MMP-2) expression in advanced ovarian serous cystoadenocarcinomas: clinical and prognostic implications.

OBJECTIVE: The object of this study was to analyse the tissue and serum metalloproteinase (MMP-2), an enzyme which degrades the basement membrane collagen type IV, as a potential marker useful in prognostic evaluation and clinical monitoring of the follow-up, in patients with advanced ovarian serous cystadenocarcinoma. MATERIALS AND METHODS: Tissue MMP-2 expression was determined in 21 FIGO stage III ovarian serous cystadenocarcinomas treated with primary surgery and adjuvant chemotherapy, and compared to 10 cystadenomas used as controls. Retrospective analysis of clinical data allowed the comparison of accepted prognostic factors to tissue MMP-2 expression for impact on disease-free survival. In fourteen out of 21 patients, serum MMP-2 levels were also analysed. RESULTS: Compared to cystadenomas, the tissue MMP-2 expression was significantly (P < 0.001) higher in serous cystadenocarcinomas. A significant relationship was observed between tissue MMP-2 and disease-free survival (P = 0.0003), independently of tumor architectural grade, lymph nodal status and residual disease after debulking surgery. Recurrence risk in patients whose carcinomas had a tissue MMP-2 > or = 29% was significantly higher than that in patients whose carcinomas demonstrated lower tissue MMP-2 expression (P = 0.004). Serum MMP-2 levels correlated with tissue staining, and also expressed a significant relationship with disease-free survival (P = 0.002). CONCLUSIONS: Tissue MMP-2 seems to be a prognostic indicator in patients with FIGO stage III ovarian serous cystadenocarcinoma, significantly correlated with recurrence risk and apparently independent of tumor architectural grade, lymph nodal status, and residual disease after debulking surgery. An interesting relationship was observed between tissue staining and MMP-2 serum levels.

Expression of 72 kilodalton type IV collagenase (gelatinase A) in benign and malignant ovarian tumors.

BACKGROUND: 72 Kilodalton (kd) type IV collagenase is a matrix metalloproteinase that specifically cleaves type IV collagen molecules. The enzyme has been postulated to have an important role in the invasion and spread of malignant tumors. EXPERIMENTAL DESIGN: In situ hybridization was used to study the expression of the 72 kd type IV collagenase mRNA in 24 benign, 2 semimalignant, and 15 malignant ovarian tumors and in 5 metastases of ovarian serous adenocarcinomas. The results were correlated with the expression of the mRNA for the alpha 1(IV) chain of type IV collagen and with the corresponding immunohistochemical distribution of the enzyme. RESULTS: The results showed that the more malignant an ovarian tumor was, the more clearly mRNA expressions for both 72 kd type IV collagenase and the alpha 1(IV) chain could be detected in tumor cells. The expression of both types of mRNAs was localized within the cells of tumor stroma and occurred mainly in fibroblasts and vascular endothelial cells. Epithelial tumor cells only rarely expressed these mRNAs. Immunohistochemical stainings localized the 72 kd collagenase as well to the stromal cells as to the epithelial cells of both benign and malignant tumors. CONCLUSIONS: The findings indicate that genes for the 72 kd type IV collagenase and for its substrate are simultaneously active in the same cells of the tumor stroma. The difference in the in situ hybridization and immunohistochemical findings could be explained by a possible variation in the metabolic balance between synthesis and accumulation of the protein in different cell types. It can also be proposed that the activity of the 72 kd type IV collagenase would be mediated through a receptor-like mechanism present on epithelial cells which could bind the 72 kd type IV collagenase synthesized elsewhere. There is also a possibility that the gelatinolytic activity of the mesenchymally synthesized 72 kd type IV collagenase would be consumed to degrade extracellular matrix proteins other than basement membranes.

Glutathione S-transferase expression in benign and malignant ovarian tumours.

Glutathione S-transferase sub-types alpha, mu and pi were assessed by immunocytochemistry in 109 biopsies of ovarian tissue, comprising malignant epithelial tissue in 86 cases and tissue of ovarian origin considered to be normal in 23. Glutathione S-transferase pi was the most prevalent, being present in all except one malignant epithelium studied and 83% of non-malignant tissue. There were no significant differences in the overall distribution of positive staining for alpha, mu and pi in the malignant and non-malignant biopsies, although the intensity of staining was greater in the malignant epithelium. Stromal staining was in general more pronounced in the malignant biopsies, and this was particularly prominent in the case of the alpha sub-type. Positive staining was seen more frequently in the less well-differentiated tumours, and a diffuse cytoplasmic pattern was the most common observation in tumours of moderate and poor differentiation. There was no significant association between survival and the presence or absence of sub-type staining of alpha and mu sub-type. For the sub-type pi, patient survival was found to correlate with the intensity of staining (on a 0-(+++) scale). Those patients showing resistance to cytotoxic chemotherapy were found to have a higher intensity of staining for GST pi than responding patients.

Cyst fluid analysis in the differential diagnosis of pancreatic cysts. A comparison of pseudocysts, serous cystadenomas, mucinous cystic neoplasms, and mucinous cystadenocarcinoma.

Pancreatic cystic lesions include inflammatory pseudocysts, benign serous tumors, and mucinous neoplasms, some of which are malignant. Clinical and radiologic indices are often inadequate to discriminate reliably among these possibilities. In an attempt to develop new preoperative diagnostic criteria to assist in decisions regarding therapy, the authors have performed cyst fluid analysis for tumor markers (carcinoembryonic antigen: CEA, CA 125, and CA 19.9), amylase content, amylase isoenzymes, relative viscosity, and cytology on 26 pancreatic cysts. The cases included nine pseudocysts, five serous cystadenomas, 4 mucinous cystic neoplasms, 7 mucinous cystadenocarcinomas, and one mucinous ductal adenocarcinoma with cystic degeneration. Carcinoembryonic antigen levels were high (> 367) in all benign and malignant mucinous cysts, but were low (< 23) in the pseudocysts and benign serous cystadenomas, an indication that CEA discriminates between mucinous and nonmucinous cysts (p < 0.0001). Values for CA 125 were high in all malignant cysts, low in pseudocysts, and variable in mucinous cystic neoplasms and serous cystadenomas. Levels of Ca 19.9 were nondiscriminatory. Cyst fluid amylase and lipase content were variable but were generally high in pseudocysts and low in cystic tumors. Amylase isoenzyme analysis was useful to differentiate pseudocysts from cystic tumors. Measurement of the relative viscosity in cyst fluid showed high (> serum viscosity) values in 89% of mucinous tumors and low values (< serum) in all pseudocysts and serous cystadenomas (p < 0.01). Cytologic analysis of cyst fluids was of limited value in differentiating pseudocysts from serous cystadenoma, but in seven of eight mucinous tumors provided useful diagnostic information and correctly classified three of five malignant tumors. The authors conclude that cyst fluid analysis can provide a preoperative classification of these diagnostically difficult lesions. The combination of viscosity, CEA, CA 125, and cytology can reliably distinguish malignant cystic tumors and potentially premalignant mucinous cystic neoplasms from pseudocysts and serous cystadenomas. Amylase content with isoenzyme analysis is useful to identify pseudocysts.

Glutathione S-transferase activity and isoenzyme composition in benign ovarian tumours, untreated malignant ovarian tumours, and malignant ovarian tumours after platinum/cyclophosphamide chemotherapy.

Glutathione S-transferase (GST) isoenzyme composition, isoenzyme quantities and enzymatic activity were investigated in benign (n = 4) ovarian tumours and malignant ovarian tumours, before (n = 20) and after (n = 16) chemotherapy. Enzymatic activity of GST in cytosols was measured by determining 1-chloro-2,4-dinitrobenzene conjugation with glutathione, cytosolic GST subunits were determined by wide pore reversed phase HPLC, using a S-hexylglutathione-agarose affinity column, and isoelectric focussing. Both GST activity and GST pi amount were not related to histopathologic type, differentiation grade, or tumour volume index in untreated malignant tumours. GST isoenzyme patterns were identical in benign tumours and malignant tumours before and after platinum/cyclophosphamide chemotherapy, while GST pi was the predominant transferase. Mean GST activity and GST pi amount were decreased (P < 0.05) in malignant ovarian tumours after platinum/cyclophosphamide chemotherapy compared to untreated ovarian malignant tumours. No relation was found in untreated ovarian tumours between GST pi amount and response to platinum/cyclophosphamide chemotherapy. Thus, within the limitations of the current study no arguments were found for a role of GST in in vivo drug resistance of malignant ovarian tumours to platinum/cyclophosphamide chemotherapy.