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Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biological understanding and biomarker development. In this study, we fractionate blood plasma by combining size-exclusion chromatography (SEC) and OptiPrep density gradient centrifugation to study clinical context-dependent and time-dependent variations in the biomolecular landscape of systemically circulating EV. Using pooled blood plasma samples from breast cancer patients, we first demonstrate the technical repeatability of blood plasma fractionation. Using serial blood plasma samples from HIV and ovarian cancer patients (n = 10) we next show that EV carry a clinical context-dependent and/or time-dependent protein and small RNA composition, including miRNA and tRNA. In addition, differential analysis of blood plasma fractions provides a catalogue of putative proteins not associated with systemically circulating EV. In conclusion, the implementation of blood plasma fractionation allows to advance the biological understanding and biomarker development of systemically circulating EV., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Background: This study aimed to detect the eukaryotic initiation factor 3B (EIF3B) expression and explore its correlation with clinical features and prognosis in epithelial ovarian cancer (EOC) patients., Methods: A total of 230 primary EOC patients underwent surgery treatment were retrospectively reviewed. Immunohistochemical (IHC) assay was used to determine EIF3B expression in tumor and adjacent tissue specimens of all patients. According to the total IHC score, the expression of EIF3B was classified as low expression and high expression, and the latter was further divided into 3 grades: high+, high++, and high+++ expressions. Overall survival (OS) was calculated., Results: Eukaryotic initiation factor 3B expression was increased in tumor tissue compared with adjacent tissue. Tumor EIF3B high expression correlated with larger tumor size (>10 cm), lymphatic metastasis, and advanced International Federation of Gynecology and Obstetrics stage (FIGO) (III/IV). Besides, OS was decreased in patients with tumor EIF3B high expression compared with patients with tumor EIF3B low expression, and further analysis showed that the OS was shortest in patients with tumor EIF3B high+++ expression, followed by patients with tumor EIF3B high++ expression and patients with tumor EIF3B high + expression, and the longest in patients with tumor EIF3B low expression. Additionally, higher tumor EIF3B expression, peritoneal cytology (positive), ascites volume (>100 mL), differentiation (poor vs. well/moderate), tumor size (>10 cm), FIGO stage (III/IV vs. I/II), and cancer antigen 125 (>1000 U/mL) independently predicted shorter OS., Conclusion: Eukaryotic initiation factor 3B exhibits a clinical value for monitoring disease progression and predicting prognosis in EOC patients., (© 2020 The Authors. Journal of Clinical Laboratory Analysis Published by Wiley Periodicals LLC.)

Background: Standard biomarker testing of a single macroscopic disease site is unlikely to be sufficient because of tumor heterogeneity. A focus on examining global biomarker expression or activity, particularly in microscopic residual chemotherapy-resistant disease, is needed for the appropriate selection of targeted therapies. This study was aimed at establishing a technique for the assessment of biomarkers of ovarian cancer peritoneal spread., Methods: An in-house developed fluorescent imaging device was used to detect the expression of the c-Met oncogene in ovarian cancer. A modified cyanine 5-tagged peptide, GE137, with a high in vitro affinity for the human c-Met protein, was tested in a panel of ovarian cancer cell lines. Finally, the feasibility of detecting submillimeter ovarian cancer cell peritoneal metastases in vivo was tested through the intravenous injection of GE137 into mice with tumor xenografts., Results: Using optical imaging it was possible to detect c-Met expression in submillimeter peritoneal metastases that were freshly excised from a human high-grade serous ovarian cancer. GE137 selectively bound to the c-Met tyrosine kinase without activating survival signaling pathways (AKT or extracellular signal-regulated kinase phosphorylation) downstream of c-Met. GE137 specifically accumulated in SKOv3 ovarian cancer cells expressing c-Met via clathrin-mediated endocytosis and emitted a fluorescent signal that lasted for at least 8 hours in tumor xenografts in vivo with a sustained high signal-to-noise ratio., Conclusions: Our results suggest that intraoperative optical imaging could provide a new paradigm for selecting cancer patients for appropriate targeted therapies, particularly after initial chemotherapy., (© 2014 American Cancer Society.)

Background: Ovarian cancer cells in malignant effusions lack attachment to solid-phase matrix substrata and receive survival stimuli through cell-cell and cell-soluble matrix molecule interactions. We hypothesized that adhesion-related survival and proliferation pathway signals can inform clinical outcomes and guide targeted therapeutics., Methods: Lysed cell pellets from a blinded set of benign (n = 20) and malignant (n = 51) peritoneal and pleural ovarian cancer patient effusions were applied to reverse-phase protein arrays and examined using validated antibodies to adhesion-associated protein endpoints. Results were subjected to hierarchical clustering for signature development. Association between specimen type, protein expression, and clinicopathologic associations were analyzed using the Mann-Whitney U test. Survival outcomes were estimated using the Kaplan-Meier method with log-rank comparison., Results: A cell adhesion protein signature obtained from unsupervised clustering distinguished malignant from benign effusions (P = 6.18E-06). Protein subset analyses from malignant cases defined 3 cell adhesion protein clusters driven by E-cadherin, epithelial cell adhesion molecule, and N-cadherin, respectively. The components of the E- and N-cadherin clusters correlated with clinical outcome by Kaplan-Meier statistics. Univariate analysis indicated that FAK and phosphorylated AKT were associated with higher overall and progression-free survival (PFS) (P = .03), and Akt, phosphorylated paxillin, and E- and N-cadherin were associated with improved PFS (P ≤ .05). If 4 or 5 of the index adhesion proteins were high, PFS was improved by multivariate analysis (P ≤ .01)., Conclusions: This hypothesis-testing examination of tumor cell adhesion molecules and pathways yielded potential predictive biomarkers with which to triage patients to selected molecular therapeutics and may serve as a platform for biomarker-based stratification for clinical application., (Copyright © 2011 American Cancer Society.)

An unassigned and prominent resonance in the region from delta 2.0-2.1 ppm has frequently been found in the in vivo MR spectra of cancer patients. We demonstrated the presence of this resonance with in vivo MRS in the cyst fluid of a patient with an ovarian tumor. (1)H-NMRS on the aspirated cyst fluid of this patient confirmed the observation. A complex of resonances was observed between 2.0 and 2.1 ppm. It was also present in 11 additional ovarian cyst fluid samples randomly chosen from our biobank. The resonance complex was significantly more prominent in samples from mucinous tumors than in samples from other histological subtypes. A macromolecule (>10 kDa) was found responsible for this complex of resonances. A correlation spectroscopy (COSY) experiment revealed cross peaks of two different types of bound sialic acid suggesting that N-glycans from glycoproteins and/or glycolipids cause this resonance complex. In the literature, plasma alpha-1 acid glycoprotein (AGP), known for its high content of N-linked glycans, has been suggested to contribute to the delta 2.0-2.1 spectral region. The AGP cyst fluid concentration did not correlate significantly with the peak height of the delta 2.0-2.1 resonance complex in our study. AGP may be partly responsible for the resonance complex but other N-acetylated glycoproteins and/or glycolipids also contribute. After deproteinization of the cyst fluid, N-acetyl-L-aspartic acid (NAA) was found to contribute significantly to the signal in this spectral region in three of the 12 samples. GC-MS independently confirmed the presence of NAA in high concentration in the three samples, which all derived from benign serous tumors. We conclude that both NAA and N-acetyl groups from glycoproteins and/or glycolipids may contribute to the delta 2.0-2.1 ppm resonance complex in ovarian cyst fluid. This spectral region seems to contain resonances from biomarkers that provide relevant clinical information on the type of ovarian tumor.

Background: The authors recently reported on the role of cyclin E in differentiating ovarian/primary peritoneal carcinoma from malignant peritoneal mesothelioma using gene expression arrays. In the current study, they analyzed the expression of low-molecular weight (LMW) forms of cyclin E in ovarian carcinoma, malignant mesothelioma, and benign reactive effusions., Methods: Cyclin E protein expression was analyzed in 98 effusions (72 ovarian carcinomas, 14 malignant mesotheliomas, and 12 reactive specimens) using immunoblotting. Sixty-two ovarian carcinoma effusions were studied further for cyclin E expression using immunohistochemistry. The correlations between cyclin E expression in ovarian carcinoma and clinical parameters, including chemotherapy response, were analyzed., Results: LMW forms of cyclin E were identified in 54 of 72 ovarian carcinoma effusions (75%) compared with 1 of 14 malignant mesothelioma effusions (7%) and 1 of 12 reactive effusions (8%) (P < .001). Their presence in ovarian carcinoma was associated with a higher percentage of cyclin E-positive cells (P = .001) and increased staining intensity (P < .001) using immunohistochemistry. The presence of LMW forms of cyclin E was correlated with shorter overall survival (P = .021) and progression-free survival (P = .020). The presence of a higher percentage of cyclin E-positive cells using immunohistochemistry was correlated with shorter progression-free survival (P = .026). No association with chemotherapy response was observed., Conclusions: LMW forms of cyclin E differentiated ovarian carcinoma from benign and malignant mesothelial cells and were associated with increased protein expression using immunohistochemistry. The expression of LMW cyclin E forms was not associated with chemotherapy response, although it may be a marker of aggressive disease in patients with metastatic ovarian carcinoma., ((c) 2007 American Cancer Society.)

Background: Cyclins, cyclin dependent kinases (cdks), and their inhibitors act in combination to regulate progression through the cell cycle and often are dysregulated in carcinoma. The authors hypothesized that cyclin E plays an important role in ovarian carcinogenesis and that its overexpression may be an indicator of a poor prognosis., Methods: Immunohistochemical analysis of cyclin E expression was performed by image analysis in normal ovaries, cystadenomas, tumors of low malignant potential, and 405 primary ovarian carcinomas by using tissue microarray technology., Results: Overexpression of cyclin E was found in 63.2% of the samples and was associated with clear cell, poorly differentiated, and serous carcinoma (P < or = .001), high-grade tumors (P < or = .001), late-stage disease (P = .002), age older than 60 years at the time of diagnosis (P = .04), and suboptimal cytoreduction (P = .001). A high percentage of cyclin E-expressing cells was associated with a poor outcome in univariate and in multivariate analyses. In addition, cyclin E levels also reduced survival in the late-stage disease group and in patients who underwent suboptimal debulking., Conclusions: Cyclin E was identified as an independent prognostic factor in patients with ovarian carcinoma. The accumulation of cyclin E protein may be a late event in tumorigenesis and may contribute to disease progression in these patients.

Background: Multiple epidemiologic and histologic studies have suggested that ovarian endometriosis can give rise to malignant ovarian tumors, primarily those of epithelial origin. The progression of endometriosis to endometriosis-associated ovarian carcinoma (EAOC) has not been investigated thoroughly and is poorly understood at best. Using immunohistochemical methods, we compared the differential expression patterns of various cytokines and growth factors in atypical endometriosis (AE) and EAOC., Methods: Using the Johns Hopkins Pathology Data Bank, tissue blocks from patients diagnosed with EAOC or AE were identified. Tissue blocks were stained for 4 markers: vascular endothelial growth factor (VEGF), Ki-67, estrogen receptor (ER), and progesterone receptor (PR)., Results: Seventeen cases of EAOC and 8 cases of AE were identified. Staining for VEGF was documented in 16 of 17 (94%) EAOC tissue blocks and in only 1 of 8 (12.5%) AE tissue blocks (P < 0.0001). Only 4 of the 17 (23%) EAOC tissue blocks exhibited positive staining for ER, compared with 8 of 8 (100%) AE tissue blocks (P = 0.0005). Positive staining for PR was noted in only 6 of 17 (35%) EAOC samples but was present in 8 of 8 (100%) AE samples (P = 0.003). Seventy percent of EAOC samples exhibited positive staining for Ki-67, compared with 37.5% of AE samples (P = 0.19)., Conclusions: EAOC appears to be associated with overexpression of VEGF and reduced expression of both ER and PR. Variations in VEGF expression may be associated with the malignant transformation of endometriosis and may present both diagnostic and therapeutic options for the treatment of ovarian malignancies., (Copyright 2003 American Cancer Society.)

Background: Tyrosine kinases, such as c-KIT, c-ABL, and platelet-derived growth factor-beta (PDGFR-beta), are important regulators of cell growth. Highly potent and selective inhibitors of tyrosine kinases are being investigated as alternatives to standard chemotherapy. One such inhibitor, imatinib mesylate, is being used to treat gastrointestinal stromal tumors and chronic myelogenous leukemia. Ovarian carcinomas frequently develop resistance to conventional chemotherapeutic agents. Immunohistochemical expression of c-ABL, PDGFR-beta, and c-KIT was evaluated in ovarian carcinomas to determine whether treatment with imatinib mesylate might be feasible., Methods: The expression of c-ABL, c-KIT, and PDGFR-beta in tumors was evaluated by immunohistochemical analysis of 52 ovarian serous carcinomas, including 21 low-grade (well differentiated) and 31 high-grade (poorly differentiated) tumors. Fourteen normal ovaries were also evaluated., Results: In normal ovarian surface epithelium, c-ABL was expressed universally. PDGFR-beta was expressed in the majority (93%) of samples of normal ovarian epithelium, whereas the c-KIT protein was undetectable in normal ovarian surface epithelium. Overall, c-ABL was expressed in 71% of serous carcinomas. c-ABL was expressed more frequently in the low-grade serous carcinomas (81%) compared with the high-grade serous carcinomas (65%). PDGFR-beta expression was observed in 81% of serous carcinomas overall and was observed more frequently in higher-grade tumors. c-KIT immunohistochemical staining was absent in low-grade tumors but was present in 26% of high-grade serous carcinomas., Conclusions: The majority of ovarian serous carcinomas express one or more of the kinases targeted by the tyrosine kinase inhibitor, imatinib mesylate, suggesting the potential usefulness of this drug in the treatment of ovarian carcinoma., (Copyright 2003 American Cancer Society.DOI 10.1002/cncr.11561)

Background: Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is a growth factor commonly used to avoid leukopenia after chemotherapy. Endogenous G-CSF is produced by macrophages and granulocytes that infiltrate tumors. It has been reported that rhG-CSF stimulates the proliferation of several cell lines as well as bladder carcinoma cells. Conversely, in some hematopoietic cell lines such as U-937, WEHI-3B, and K-562 no effect or in some cases a differentiation pattern was found. Moreover, the role of rhG-CSF on the proliferation of solid tumors is not well understood., Methods: In this study, 10 ovarian carcinoma biopsies were characterized for the presence of G-CSF and G-CSF receptor by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical analysis. Proliferation was analyzed by ATP viability assays., Results: Performing RT-PCR, these biopsies and four ovarian carcinoma cell lines were analyzed for endogenous G-CSF production, which was found in some biopsies and in all cell lines. Despite the presence of the G-CSF receptor in all biopsies and cell lines, no proliferation was found after rhG-CSF incubation of the cell lines or the tumor samples for 3 and for 6 days, respectively., Conclusions: Summarizing the authors' in vitro studies, rhG-CSF does not affect the proliferation of ovarian carcinoma cells in vitro., (Copyright 2001 American Cancer Society.)

Background: The purpose of the current study was to determine vascular endothelial growth factor (VEGF) concentrations in cyst fluid from malignant, borderline, and benign ovarian tumors, and to correlate these data with preoperative serum VEGF concentrations and clinicopathologic characteristics., Methods: One hundred seven ovarian cysts were removed and punctured for cyst fluid collection. Histologic diagnosis revealed 25 malignant, 12 borderline, and 70 benign ovarian tumors. The VEGF concentrations of all the cyst fluid specimens as well as in 37 preoperatively collected serum samples were determined by making use of a sandwich type double determinant enzyme linked immunoadsorbent assay based on a combination of 4 polyclonal antibodies., Results: Statistically significantly higher VEGF concentrations were found in cyst fluid from malignant (median, 21.5 microg/L) compared with borderline (median, 3.2 microg/L; P = 0.01) or benign tumors (median, 1.3 microg/L; P < 0.0001). Preoperative serum VEGF concentrations were significantly higher in patients with malignant (median, 0.63 microg/L; range, 0.016-17.7 microg/L) compared with nonmalignant tumors (median, 0.28 microg/L; range, 0.016-0.89 microg/L; P = 0.008). A significant correlation of preoperative serum VEGF was found with VEGF cyst fluid concentrations (r = 0.38; P = 0.02). Significantly higher VEGF cyst fluid concentrations were found in serous malignant (median, 31.9 microg/L) compared with mucinous malignant tumors (median, 4.7 microg/L; P = 0.004). Not significant, though higher median VEGF cyst fluid concentrations were found in advanced International Federation of Gynecology and Obstetrics (FIGO) Stage II, III, and IV, histologic Grade 2 and 3, patients with residual tumor greater than 2 cm, with malignant cells in ascites or peritoneal washings, or with recurrent disease, as compared with FIGO Stage I, histologic grade 1, patients with less than or equal to 2-cm residual tumor, without malignant cells in ascites/peritoneal washings, or without recurrent disease, respectively., Conclusions: It has become clear from the increased study sample that ovarian tumors of different histologic etiology vary in VEGF cyst fluid concentrations, with the highest VEGF cyst fluid concentrations in malignant tumors. The prognostic significance of VEGF cyst fluid concentrations in advanced FIGO stage seems to be of limited value but may be important in the selection of high risk FIGO Stage I and borderline types. Data from this study indicate a possible role for VEGF as a serum tumor marker.

Background: The presence of malignant cells in peritoneal washings is an independent prognostic factor in the evaluation of gynecologic malignancies. The differentiation between reactive mesothelial cells and carcinoma cells can be a diagnostic challenge based on morphology alone. The expression of some cell adhesion molecules may be helpful in the differential diagnosis., Methods: To evaluate the specificity of 2 transmembrane cell adhesion proteins (E-cadherin and CD44) in the differentiation of mesothelial cells from carcinoma cells in pelvic washings, formalin fixed, paraffin embedded cell blocks of pelvic washings from 19 cases of metastatic ovarian adenocarcinoma and 16 cases of benign peritoneal washings were immunostained with monoclonal antibodies to E-cadherin and CD44. The staining patterns were evaluated blindly by three observers. Positive staining was defined as uniform membranous staining for each marker., Results: Fourteen benign peritoneal washings (87.5%) demonstrated immunoreactivity with anti-CD44. On the contrary, only four adenocarcinomas (21.1%) demonstrated anti-CD44 immunoreactivity. E-cadherin expression was identified in only 2 benign peritoneal washings (12.5%) whereas 16 adenocarcinomas (84.2%) strongly expressed E-cadherin. The differences in immunostaining for both CD44 and E-cadherin between benign and malignant peritoneal washings were statistically significant. The combination of positive staining for E-cadherin and negative staining for CD44 was 100% specific for metastatic adenocarcinoma, whereas a combination of negative staining for E-cadherin and positive staining for CD44 was 100% specific for reactive mesothelial cells., Conclusions: Both E-cadherin and CD44 reliably distinguish reactive mesothelial cells from adenocarcinoma. The combination of E-cadherin/CD44 is highly specific and is a useful diagnostic adjunct with which to distinguish benign reactive mesothelial cells from adenocarcinoma in pelvic washings.

Background: Calretinin (CR) is a valuable marker in the immunohistochemical distinction between malignant mesothelioma (MM) and adenocarcinoma (ACA) in tissue sections. However, there is limited and conflicting data regarding the utility of CR in this differential diagnosis on cytologic material, especially cell block preparations. Also, the possible role of CR in the distinction of papillary serous borderline tumor (SBT) cells from reactive mesothelial cells in peritoneal washings has not been examined., Methods: Formalin-fixed paraffin-embedded cell block specimens of cytologic fluids, washings, and aspirates with a suspicious or positive cytologic diagnosis and a confirmed diagnosis of MM (29 cases), ACA (39 cases), and SBT (10 cases) were used for CR immunohistochemistry (IHC)., Results: Moderate to strong staining in > 50% of tumor cells was noted in 26 of 29 (90%) MMs but in only 3 of 39 (8%) ACAs and 1 of 10 (10%) SBTs. Admixed reactive mesothelial cells (when present) were strongly positive in all fluid specimens, but the staining pattern of benign non-reactive mesothelial cells in washing specimens was less reliable., Conclusions: CR is both a sensitive and specific marker of reactive and neoplastic mesothelial cells in cytologic cell block preparations and is thus useful in the differential diagnosis of MM and metastatic ACA in malignant effusions. However, CR IHC does not appear to allow definitive identification of SBT in peritoneal washings.

Most ovarian tumors are cystic structures containing variable amounts of fluid. Several studies of ovarian cyst fluid focus on one specific metabolite using conventional assay systems. We examined the potential of (1)H-nuclear magnetic resonance spectroscopy in evaluation of the overall metabolic composition of cyst fluid from different ovarian tumors. Ovarian cyst fluid samples obtained from 40 patients with a primary ovarian tumor (12 malignant and 28 benign) were examined. After deproteinization and pD standardization, we performed (1)H-NMR spectroscopy on a 600 MHz instrument. With (1)H-NMR spectroscopy we found detectable concentrations of 36 metabolites with high intersample variation. A number of unassigned resonances as well as unexpected metabolites were found. We introduce an overall inventory of the low-molecular-weight metabolites in ovarian cyst fluid with corresponding resonances. Significant differences in concentration (p < 0.01) were found for several metabolites (including an unknown metabolite) between malignant and benign ovarian cysts. Furthermore, higher concentrations in malignant- and lower in benign fluids were found compared to normal serum values, indicating local cyst wall metabolic processes in case of malignant transformation. We conclude that (1)H-nuclear magnetic resonance spectroscopy can give an overview of low-molecular-weight proton-containing metabolities present in ovarian cyst fluid samples. The metabolic composition of cyst fluid differs significantly between benign and malignant ovarian tumors. Furthermore, differences between benign subgroups possibly related to histopathological behaviour can be detected. The presence of N-acetyl aspartic acid and 5-oxoproline exclusively in serous cystadenoma samples is remarkable. Future studies will concentrate on these findings and explore the possibilities of extrapolating information from the in vitro studies to in vivo practice, in which metabolic differences between malignant and benign subtypes can be of great importance in a pre-operative phase., (Copyright 2000 John Wiley & Sons, Ltd.)

Background: Nuclear expression of Y box-binding protein (YB-1), a member of the DNA binding protein family, has been reported to be much more highly concentrated in cisplatin-resistant cell lines than in their parental counterparts, suggesting an ability to limit cisplatin sensitivity. Moreover, YB-1 plays a key role in P-glycoprotein expression. Because ovarian carcinoma traditionally has been treated with cisplatin-based chemotherapy, the sensitivity of the tumors to chemotherapy could reflect a particular prognosis in patients with ovarian carcinoma. The aim of the current study was to determine whether YB-1 expression correlated with prognosis in ovarian serous adenocarcinoma patients., Methods: The expression of YB-1 in the nucleus was examined immunohistochemically in 42 paraffin embedded primary Stage III (International Federation of Gynecology and Obstetrics) serous ovarian carcinoma tumors extirpated by primary surgery at Kyushu University Hospital between 1985-1995., Results: Of the 40 primary ovarian tumors examined, 12 (30%) were positive for YB-1 expression in the nucleus. There was no significant difference in intraperitoneal stage, histologic grade, or residual tumor size after primary surgery between patients with tumors with positive and those with negative nuclear expression of YB-1 protein. The disease free survival curve for patients whose tumors were positive for nuclear expression of YB-1 protein was significantly worse than that for patients whose tumors were negative (P = 0.0025). P-glycoprotein was overexpressed in 4 of 12 tumors with nuclear YB-1 expression (33%) but there was no statistical significance between the expression of nuclear YB-1 and P-glycoprotein., Conclusions: The expression of YB- 1 protein in the nucleus may be considered a useful prognostic marker and also may reflect the sensitivity of ovarian serous adenocarcinoma to chemotherapy.

Background: The aim of the current study was to investigate the expression of vascular endothelial growth factor (VEGF) by neoplastic cells in patients with serous ovarian tumors. The correlation between neoangiogenesis and 72-kilodalton metalloproteinase (MMP2) immunostaining also was evaluated., Methods: Fifty-eight patients with serous ovarian tumors who were treated at the Institute of Gynecology and Obstetrics of Ancona University (Ancona, Italy) were used as the study population; 10 women had serous cystoadenoma, 16 women had a serous borderline tumor, and 32 women had invasive cystoadenocarcinoma. Expression of VEGF and MMP2 was evaluated immunohistochemically by polyclonal antibody anti-VEGF (dilution, 1:100) and affinity purified, rabbit anti-MMP2, formalin fixed, paraffin embedded tissue. Positive staining was expressed as a percentage of positive cells per 10(3) counted neoplastic cells., Results: Compared with cystoadenomas and borderline tumors, the tessutal VEGF immunostaining was significantly higher in cystoadenocarcinomas, with the highest values detected in architectural International Federation of Gynecology and Obstetrics Grade 3 neoplasms (P2 < 0.001). A direct relation was observed between VEGF and MMP2 immunostaining (correlation coefficient, 0.44; P2 = 0.013). A relation was found between VEGF expression and disease free survival as evaluated by Cox hazards analysis (P2 = 0.03)., Conclusions: Neoangiogenesis detected by VEGF immunostaining appears to be a promising indicator of aggressiveness in serous ovarian tumors. In cystoadenocarcinomas, VEGF expression appears to be related to MMP2 index.

Background: Insulin-like growth factor-2 (IGF2) and H19 are reciprocally imprinted genes on chromosome 11; IGF2 is expressed paternally and H19 is expressed maternally. Loss of imprinting (LOI) at both H19 and IGF2 has been reported in seven fully informative adult testicular germ cell tumors (GCTs) and may contribute to germ cell carcinogenesis., Methods: Genomic DNA from 61 pediatric GCTs was amplified by polymerase chain reaction (PCR) and screened for heterozygosity at both IGF2 and H19 using either ApaI or RsaI, respectively. If heterozygous, polyadenylated RNA was isolated and reversed-transcribed into cDNA. cDNA then was amplified by PCR and the products were digested with restriction enzymes to evaluate GCT expression of IGF2 and H19., Results: Eleven pediatric GCTs were fully informative for H19 and IGF2, including 5 ovarian GCTs, 2 testicular GCTs, and 4 extragonadal GCTs. Consistent with prior studies, both testicular GCTs showed LOI at both H19 and IGF2. In contrast, three of the five ovarian GCTs had LOI at both IGF2 and H19; one had LOI at IGF2 only, and one retained imprinting at both loci. Only one of the four extragonadal GCTs had LOI at IGF2 whereas three of the four had LOI at H19., Conclusions: These data suggest that LOI at H19 and IGF2 also may be common in pediatric testicular GCTs. However, ovarian and extragonadal pediatric GCTs showed variable patterns of LOI that may indicate differences in the timing of carcinogenesis in germ cells at these sites.

Background: Clinical data clearly indicate a correlation between tumor neovascularization, aggressiveness of tumor growth, and metastatic spread. One of the key factors capable of stimulating tumor angiogenesis is vascular endothelial growth factor (VEGF). Using an immunoassay for VEGF, we assessed the levels of soluble VEGF in the sera and effusions of patients with malignant and nonmalignant disease as well as in the sera of healthy controls., Methods: Using a sandwich enzyme-linked immunoadsorbent assay, the concentration of VEGF was measured in serum specimens (n=445) and effusions (n=56) collected from a total of 212 patients with various types of cancer, 88 patients with nonmalignant disease, and 145 healthy individuals., Results: Low and rather stable levels of VEGF were detected in the serum of healthy individuals (median, 294 pg/mL; range, 30-1752 pg/mL; 95th percentile, 883 pg/mL). Compared with healthy individuals, serum levels in patients with acute infections were elevated (P=0.03), whereas patients with chronic cirrhosis had lower serum VEGF levels. Among patients with various types of neoplasia, VEGF serum levels in patients with ovarian or gastrointestinal carcinoma were significantly higher than in healthy individuals. Moreover, VEGF concentrations in sera from patients with metastatic disease were higher than in sera from patients with localized tumors. Maximum serum concentrations of VEGF (median, 1022 pg/mL; range, 349-7821 pg/mL) were found in patients with metastatic ovarian carcinoma. Median VEGF levels (and ranges) in malignant effusions were up to 10-fold higher than in matched serum samples: 5528 pg/mL (468-49269 pg/mL) in ovarian carcinoma, 885 pg/mL (77-14,337 pg/mL) in breast carcinoma, and 813 pg/mL (372-18,331 pg/mL) in gastrointestinal carcinoma. In contrast, ascitic fluid from patients with cirrhosis contained only 303 pg/mL (median, range 116-676 pg/mL) of VEGF, corresponding to the low serum levels in this patient group., Conclusions: Depending on the tumor type, elevated levels of VEGF are detectable in the serum of only 0-20% of patients with localized cancer but in 11-65% of patients with metastatic cancer. Of cytology-proven malignant ascites or peritoneal exudates from various malignancies, 46-96% show VEGF levels above the upper limit (95th percentile, 676 pg/mL) of nonmalignant ascites. Maximum VEGF concentrations in malignant effusions indicate abundant local release of VEGF within the pleural or peritoneal cavity. These results suggest that VEGF might play an important role in tumor progression and the formation of malignant effusions. Further studies are warranted to determine the clinical value of soluble VEGF as a tumor marker, a prognostic factor, and a surrogate indicator of tumor angiogenesis.

Background: The mechanisms by which metastatic ovarian cancer adheres to peritoneal surfaces are not well understood. A role for tumor-derived extracellular matrix adhesive molecules such as fibronectin (FN) has been proposed. Because oncofetal fibronectin (onfFN) isoforms function in the adhesion of trophoblasts and have been identified in association with several malignancies, we sought to study onfFN in patients with advanced epithelial ovarian cancer., Methods: Total FN was identified with the nonspecific anti-FN monoclonal antibody CAF. OnfFN was identified using the specific monoclonal antibodies FDC-6 and X18A4. These antibodies were applied to: 1) ascitic fluid from advanced epithelial ovarian cancer patients and peritoneal fluid from patients without pathologic conditions and 2) tissue sections of primary lesions and metastatic ovarian cancer implants. Comparative histologic specimens included normal ovarian tissue and small bowel implants of endometriosis., Results: When measured by sandwich enzyme-linked immunoadsorbent assay, all peritoneal fluids (32 malignant and 32 benign) contained marked quantities of total (CAF reactive) FN, although malignant ascites had higher concentrations than benign samples (173.2 +/- 36.8 microg/mL vs. 76.4 +/- 31.8 microg/mL; P = 0.001). Malignant ascites also had significantly higher levels of onfFN than benign peritoneal fluid (FDC-6: 3.4 +/- 0.6 vs. 0.9 +/- 0.2 microg/mL; and X18A4: 5.1 +/- 1.3 vs. 1.1 +/- 0.4 microg/mL; P = 0.0001). Immunohistochemical staining of malignant lesions revealed prominent localization of both CAF reactive FN and onfFN to the stroma surrounding epithelial tumor nests. More delicate fibrillar staining within tumor nests also was evident. In contrast, implants of endometriosis revealed strong stromal staining for CAF reactive FN but not for onfFN., Conclusions: These results demonstrate the presence of onfFN in advanced ovarian malignancies. We speculate that onfFN may participate in tumor-associated peritoneal adhesive interactions.

Background: Although the ascites of patients with ovarian carcinoma has been reported to contain immunosuppressive factors, the identity and source of this activity has not been fully elucidated. The objective of this study was to describe the purification of a single immunosuppressive protein, alpha-1 acid glycoprotein, from ovarian carcinoma ascites, identify its site of production, and describe a possible mechanism by which it inhibits lymphocytes., Methods: Ascites from proteins from five patients with epithelial ovarian carcinoma first were differentially precipitated by size with different concentrations of polyethylene glycol and then separated on the basis of isoelectric focusing. The protein factions then were placed in a lymphocyte proliferation assay to determine immunosuppressive activity. Western blot analysis was used to identify alpha-1 acid glycoprotein as an immunosuppressive protein in ascites. Total RNA was extracted from ovarian and hepatic cell lines as well as primary and recurrent ovarian tumor samples. Reverse-transcriptase polymerase chain reaction then was utilized to identify the site of production of this protein. Purified alpha-1 acid glycoprotein was placed in lymphocyte culture and its effects on lymphocyte interleukin-2 (IL-2) production were measured by enzyme-linked immunoadsorbent assay., Results: Addition of purified alpha-1 acid glycoprotein to the lymphocyte assay resulted in a 60% decrease in lymphocyte proliferation (P < 0.05). Alpha-1 acid glycoprotein transcript was not identified in ovarian tumor cells. The addition of purified alpha-1 acid glycoprotein to the lymphocyte culture resulted in a 65% decrease in IL-2 secretion into the media (P < 0.05)., Conclusions: Alpha-1 acid glycoprotein is an immunosuppressive protein purified from ovarian carcinoma ascites. It is not expressed primarily by ovarian carcinoma cells. It appears to inhibit IL-2 secretion by lymphocytes.

Background: Mucinous tumors of the ovary have been thought to originate in two ways: by müllerian-type metaplasia of surface epithelium, and as monodermal teratomas. To gain a better understanding of their pathogenesis, the authors analyzed these tumors for their expression of estrogen receptors (ER) and progesterone receptors (PR) as markers of müllerian-type differentiation and for their content of gastric-type mucin as a marker of gastric differentiation., Methods: The histochemical expression of ER, PR, and gastric-type mucin was studied in 10 specimens of the cervix with normal endocervical glands (as a representative of müllerian-derived mucin-containing cells), 3 ovary specimens with surface epithelial inclusion cysts that contained endocervical-like mucin-containing cells (representing müllerian-type metaplasia), and 47 mucinous tumors of the ovary (29 benign, 8 with low malignant potential, and 10 malignant)., Results: Normal endocervical glands expressed ER and PR and rarely expressed gastric-type mucin. Ovarian inclusion cysts showed strong expression of ER and PR in the cuboidal cells and drastically reduced expression in the endocervical-like mucin-containing cells. The cuboidal cells were negative for gastric-type mucin, but the endocervical-like mucin-containing cells expressed gastric-type mucin. Endocervical-like mucinous cells in benign and borderline mucinous tumors showed expression of PR and/or gastric-type mucin in all cases., Conclusions: The staining results for the inclusion cysts support the thesis that the endocervical-like mucinous cells encountered in the ones that express ER and PR weakly or not at all and have histochemical properties of normal gastric epithelium have their origin in metaplasia of müllerian-type epithelium. Application of the same staining methods to benign ovarian tumors and those with low malignant potential suggests strongly that similar müllerian-type metaplasia is a major pathway in their pathogenesis.

Background: The clinical significance of p53 overexpression in patients with ovarian carcinoma is uncertain. Previous studies have yielded conflicting results and have been hampered by small patient populations, failure to account for other well-known prognostic variables in multivariate analysis, and failure to account for the grade of p53 overexpression. The aim of this study was to investigate the independent prognostic significance of p53 overexpression in patients with primary ovarian epithelial cancer (POEC)., Methods: Tumors obtained from 221 patients with primary ovarian epithelial cancer (POEC) (Stages I-IV) were studied for p53 overexpression semiquantitatively by immunohistochemical techniques. The median duration of follow-up of surviving patients was 7 years. The presence or absence and degree of p53 overexpression were correlated with the clinicopathologic features of the study population and overall survival. Survival curves were constructed according to the Kaplan-Meier method, and differences in survival were assessed with the log rank test. The prognostic significance of p53 overexpression for survival was assessed in a multivariate analysis with the Cox proportional hazards model., Results: One hundred seven tumors (48.4%) exhibited p53 overexpression. The overexpression was graded as mild in 16.7% of cases, moderate in 5.9%, and strong in 25.8%. p53 overexpression was associated with advanced stage (P = 0.04), higher grade (P = 0.0003), serous histology (P = 0.0018), and patient age > 61 years (P = 0.013). In univariate analysis, p53 overexpression was a significant prognostic factor (P = 0.049 for any degree of overexpression, P = 0.03 for strong overexpression). However, in multivariate analysis, after adjustment for stage and size of residual tumor following cytoreductive surgery, p53 overexpression did not retain statistical significance. Survival curves for patients with different stages and grades of tumor differentiation did not demonstrate a difference in survival among patients with no p53 overexpression, compared with those who demonstrated any degree of p53 overexpression or compared with those who demonstrated strong p53 overexpression., Conclusions: p53 overexpression is not an independent prognostic factor for patients with primary ovarian epithelial cancer.

Background: The study was designed to improve the discrimination between functional and neoplastic ovarian cysts in order to avoid unnecessary surgery., Methods: Concentrations of tumor markers (CA 125, CEA, CASA, CA 72-4) and hormones (estradiol, FSH, LH) in cyst fluid were detected by enzyme immuno- or immunoradiometric assays. Wilcoxon test was used to evaluate the correlation of cyst fluid markers and histology., Results: One hundred and thirty-eight ovarian cyst aspirates were investigated. Seventy-one patients (51.5%) had functional cysts whereas 67 (48.5%) had benign (n=59) or malignant (n=8) cystic tumors. Statistically significant correlations of CA 125 (p<0.0005) and CASA (p<0.02) with neoplastic histology were found. No significant correlation could be detected between CA 72-4, CEA, or hormone values and histology. Elevated estradiol concentrations are suspicious for functional cysts in premenopausal age. Low FSH and LH levels seem to be an indicator for functional cysts in peri- and postmenopausal age., Conclusions: The assessed analytes could not reliably distinguish between functional and neoplastic ovarian cysts. Our results indicate that CA 125 is a marker for neoplastic histology in a proportion of ovarian cysts. The use of FSH and LH in the diagnosis of postmenopausal blastomas needs further investigation.

Objective: To study the expression of Cathepsin D (Cath D) and CA 125 antigens and ER and PR receptors on freshly obtained surgical specimens of ovarian carcinomas and their relationship with menopausal status, tumor histology, primary tumor size and lymph node invasion., Methods: The tumors obtained from 100 women were measured and cut in half. The cut surface of one half was pressed against glass slides which were air dried and stained using the Avidin-Biotin peroxidase method for Cath D and CA 125 antigens. The slides were viewed under the light microscope for the characteristic brown granules in the cytoplasm or membrane of the malignant cells. The other half of the tumor was subjected to routine histological examination and part used for the demonstration of ER and PR receptors. The results were analyzed using chi 2 analysis., Results: Cath D positivity was as common as CA 125 positivity. Cath D positivity is more frequently associated with serous carcinomas than with others. No relationship was observed between ER/PR positivity and Cath D or CA 125 positivity., Conclusion: The high incidence of Cath D positivity makes it a possible complementary method for following up ovarian carcinoma patients especially those who are CA 125 negative.

Background: Tumors of borderline malignancy are still a controversial subgroup of ovarian neoplasms. The expression of several cell regulatory proteins was studied to characterize the molecular phenotype of these tumors, and to compare them with their benign and malignant counterparts., Methods: Specimens from 22 patients with tumors of borderline malignancy (11 serous and 11 mucinous tumors), 12 patients with benign tumors, and 16 patients with invasive ovarian carcinomas were evaluated for expression of epidermal growth factor receptor (EGFR), HER-2/neu, PTP1B, and p53 by immunohistochemical techniques., Results: One or both of the tyrosine kinase growth factor receptors EGFR and HER-2/neu was expressed by 42% of benign, 59% of borderline, and 81% of malignant ovarian tumors. EGFR was expressed in a significantly greater fraction of malignant lesions (69%) than borderline lesions (18%) (P< 0.004). EGFR expression was not observed among the 11 mucinous borderline tumors. HER-2/neu was expressed by 50% of borderline tumors and was not a marker for malignancy. The tyrosine phosphatase PTP1B was expressed by a similar fraction of benign (17%), borderline (27%), and malignant (19%) tumors. The number of cases studied precluded correlation of kinase and phosphatase activity. However, among 12 tumors with PTP1B expression, 9 also expressed EGFR or HER-2/neu. Overexpression of p53 was observed only in malignant serous tumors and was not found in malignant mucinous, borderline, or benign lesions., Conclusions: Either EGFR or HER-2/neu was detected in a majority of borderline cancers. PTP1B was present only in a minority of these cancers. Frankly malignant serous lesions differed from borderline and benign tumors with regard to expression of EGFR and overexpression of p53.

Background: A large number of monoclonal antibodies (MoAbs) against human tumor cells have been generated and it has been shown that these MoAbs are useful tools in the diagnosis and treatment of cancer patients, as well as in the basic investigation of the oncogenesis and characterization of cancer cells., Methods: The 22-1-1 MoAb was established by cell fusion between mouse myeloma cells and spleen cells derived from mice immunized with the human uterine cervical adenocarcinoma cell line, SiSo. The tissue distribution and biologic characteristics of the 22-1-1 antigen (Ag) were examined., Results: The 22-1-1 Ag was distinct from the known tumor-associated antigens such as YH 206, GA 733, CA 125, carcinoembryonic antigen, and sialyl Le(x) molecules in an expression pattern in human tumor cell lines. An immunohistochemical study revealed that 22-1-1 Ag was expressed in 87.5% of uterine cervical adenocarcinomas, 66% of uterine endometrial adenocarcinomas, and 58.8% of ovarian carcinomas. Moreover, 22-1-1 Ag was detected in 87.7% of uterine cervical squamous cell carcinomas; however, it was not detected in 87.7% of uterine cervical or ovarian tissues, except in uterine endometrial glands, in which its expression was observed at low levels. The 22-1-1 Ag was secreted into cell culture supernatant fluids and was also detected in the vaginal discharges of uterine cervical carcinoma patients. The antigenic epitope of 22-1-1 Ag was shown to be a protein with a molecular weight of 78 kilodaltons using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis., Conclusions: The 22-1-1 MoAb reactive to a novel tumor-associated antigen was generated. This Ag was expressed in cancer cells derived mainly from the uterus and ovary. Moreover, 22-1-1 Ag was associated in the vaginal discharges of uterine cervical carcinoma patients. 22-1-1 MoAb is a potential tool for the study of oncogenesis and the management of cancer patients.

Human chorionic gonadotropin (hCG)-like molecules have been reported to be elevated in a substantial fraction of serum samples from patients with various gynaecologic tumours and have been discussed as possible markers in these malignancies. Employing highly sensitive and specific immunoradiometric assays, we determined total hCG-related immunoreactivity (hCG/hCG beta), as well as free alpha-subunit (alpha-SU), common to all glycoprotein hormones, in serum (n = 106) and malignant effusions (n = 26) of women with gynaecologic malignancies. For comparison, we also measured hCG/hCG beta in nonmalignant ascitic fluids (n = 21). HCG/hCG beta serum levels were elevated (> 5 IU L-1) in 39 of 106 patients (37%) with gynaecologic malignancies, whereas free alpha-SU was above normal range only in seven (6.6%). Frequencies of hCG/hCG beta elevations were similar in women with endometrial, (n = 39), cervical (n = 40) and ovarian (n = 27) cancer, being 30%, 35% and 41%, respectively. In malignant ascites (n = 15) and tumour cyst fluids (n = 11) of patients with ovarian cancer, hCG/hCG beta concentrations were significantly higher than in the corresponding serum samples and benign ascitic samples. Free alpha-SU, on the other hand, was increased in only one of 26 malignant effusions. In conclusion, hCG/hCG beta is frequently elevated in serum of patients with endometrial, cervical and ovarian cancer and may serve as a tumour marker in these malignancies, particularly in patients where other markers are negative. In this respect, analysis of ascitic or tumour cyst fluids may be of higher diagnostic value as serum measurements.

Background: The clinical significance of p53 suppressor gene nucleoprotein immunostaining in ovarian epithelial cancer has not been determined., Methods: p53 protein expression was studied by immunohistochemistry from paraffin embedded tissue in a series of 136 patients with malignant ovarian epithelial tumors. The median follow-up time of the patients still alive was 10 years., Results: Sixty (44%) carcinomas stained clearly positive for p53 protein. Positive staining for p53 protein was associated with the serous histologic type (P = 0.0006), a higher than the median S-phase fraction size determined by DNA flow cytometry (P = 0.02), and poor histologic grade of differentiation (P = 0.04), but not with the International Federation of Gynecology and Obstetrics (FIGO) stage, age at diagnosis, or DNA ploidy. Cancers with positive staining had only 17% 5-year and 9% 15-year survival rates compared with 42% 5-year and 36% 15-year survival rates corrected for intercurrent deaths among the rest of patients (P = 0.002). In a multivariate analysis, positive p53 staining was associated with poor survival (relative risk of death, 1.8, 95% confidence interval [CI], 1.2-2.9) together with less than radical surgery (nonradical vs. radical: RR, 5.5; 95% CI, 2.2-13.6), and advanced FIGO stage (RR, 1.4; 95% CI, 1.0-2.0)., Conclusion: Although p53 protein immunostaining is associated with several other prognostic factors in epithelial ovarian cancer, it may also have independent prognostic value in this disease.

Background: Papillary serous carcinoma of the peritoneum (PSCP) is a primary peritoneal tumor in women that histologically resembles papillary serous carcinoma of the ovary (PSCO). Recognition of PSCP as an entity is controversial, as is the histogenesis, histopathologic differential diagnosis, and treatment., Methods: Ten cases of PSCP retrieved from the pathology files of 4 hospitals in Kyoto and Nara, Japan, were studied clinicopathologically and immunohistochemically., Results: Patient ages at presentation ranged from 40 to 74 years (median, 56 years). All patients were Asian (Japanese). None of the patients had a history of asbestos exposure. Most of the patients had abdominal swelling, ascites with positive cytology, and elevated serum CA125. At surgery, omental tumors with dissemination to the abdominal and pelvic peritoneum were found in all patients. The histology was similar to that of Grade 2 to 3 PSCO. Immunohistochemical studies using a panel of monoclonal antibodies against carbohydrates showed that Lewis Y is a good marker, in addition to S-100, placental alkaline phosphatase, CA125, and CD15 for separating PSCP from malignant mesothelioma (MM). With cytoreductive surgery and cisplatin-based combination chemotherapy and in some cases adoptive immunotherapy and radiation, a median survival of 27 months and a 5-year survival rate of 27% were attained. One patient with Grade 3 tumor has survived for more than 6 years after surgery., Conclusions: (1) Papillary serous carcinoma of the peritoneum is a definite clinicopathologic entity; (2) immunohistochemistry is a useful tool for distinguishing PSCP from MM; (3) cytoreductive surgery and cisplatin-based combination chemotherapy with other adjunct therapies such as immunotherapy and radiation may improve patient survival in PSCP.

Background: Strong correlations have been reported between expression of urokinase-type plasminogen activator (uPA) and poor prognosis in several human carcinomas. The clinical significance of secreted levels of uPA, its receptor (uPAR), and its inhibitors (PAI-1 and PAI-2) in the ascites of patients with epithelial ovarian cancer patients was investigated., Methods: uPA, uPAR, PAI-1, and PAI-2 were measured in the primary ascites of 36 patients with epithelial ovarian cancer by enzyme-linked immunosorbent assay, and normalized to protein content. The relationship between levels of these factors in ascites and clinicopathologic variables, survival, and disease free interval (DFI) was studied. Because high levels of macrophage colony stimulating factor (CSF-1) in ascites is a known poor prognostic indicator for ovarian cancer, the effect of CSF-1 on secretion of PAI-2 by ovarian cancer cells was also examined in vitro., Results: High uPA levels per se did not correlate with either survival or DFI. However, the ratio of uPAR normalized for uPA content correlated inversely with FIGO stage and residual disease, with a significant association between high uPAR/uPA level and prolonged survival and DFI. High PAI-1 levels (> 0.120 ng/mg) proved to be a good prognostic factor, correlating with both prolonged DFI and residual disease < or = 5 cm among Stage 3 patients. Conversely, high levels of PAI-2 (> 0.061 ng/mg) indicated a poor prognosis in Stage 3 patients, as high PAI-2 levels were associated with shorter DFI and survival. CSF-1 stimulated the secretion of PAI-2 by 2.4-fold in Bix3 ovarian carcinoma cells. Multivariate analysis revealed that the two independent prognostic factors for DFI in Stage 3 patients were PAI-1 and PAI-2; Stage 3 patients with high secreted PAI-1 and low PAI-2 levels have a median DFI that was prolonged by 30 months relative to the rest of the group., Conclusion: Secreted uPAR/uPA appears to measure tumor burden and predicts prolonged DFI and survival, but was not found to be an independent prognostic factor on multivariate analysis. High levels of PAI-1 in ascites were independently associated with prolonged DFI among Stage 3 patients. Therefore, secreted uPAR and PAI-1 may modulate uPA activity and lead to improved outcome. This contrasts with the finding that secreted PAI-2 is an independent factor indicating poor prognosis, which may relate in part to the actions of CSF-1. This study emphasizes the importance of uPA, its receptor, and its inhibitors in patients with epithelial ovarian cancer.

Background: It has been shown that the p53 gene is mutated in 30-80% of ovarian carcinomas and that the genetic alterations most often manifest as an accumulation of mutant p53 protein in tumor tissue. The prognostic significance of these findings for patients with ovarian cancer, however, must be established clearly., Methods: Mutant p53 protein in 90 consecutive epithelial ovarian carcinomas was quantitatively analyzed using a time-resolved immunofluorometric procedure. In contrast to immunohistochemical techniques, this method uses two anti-p53 antibodies. The Cox model was used to evaluate the strength of the associations between the prognostic markers and disease relapse or death at univariate and multivariate levels. Kaplan-Meier survival curves were calculated for patients who were p53-positive or negative and for subgroups with a different clinical stage, histologic grade, or residual postsurgical tumor., Results: The positivity rates for p53 included 1/21 (5%) with Stage I disease, 1/6 (17%) with Stage II, 29/51 (57%) with Stage III, and 8/12 (67%) with Stage IV (total = 39/90, 43%). Patients with p53-negative tumors had a significantly longer disease free survival than did patients with p53-positive tumors (P = 0.03); these results were similar for overall survival (P = 0.06). Multivariate analysis revealed that the presence of postsurgical residual tumor was the only predictor significantly associated with poor patient outcome. However, when patients were divided into groups based on histologic grade, patients with well (G1) and moderately (G2) differentiated tumors had a significantly higher risk of cancer relapse and death if mutant p53 protein was present in their tumors compared with patients who were negative for mutant p53 protein (< 0.01)., Conclusions: The immunofluorometric measurement of mutant p53 protein accumulation in epithelial ovarian carcinomas of a low histologic grade was associated significantly with an increased risk for cancer relapse and death. A similar trend also was suggested for early stage disease and in the absence of residual tumor after surgery. These increased risks, however, were not found for patients with high grade or advanced stage cancer or for those with residual tumor. To the authors' knowledge, this is the first report suggesting that p53 tumor protein accumulation is a marker of poor prognosis in a subset of patients with ovarian cancer.

Background: Type I collagen is a major constituent of the interstitial connective tissue. Although ovarian carcinoma is known to induce the expression of type I collagen in the peritoneal cavity, the distribution and metabolic activity of this collagen in ovarian tumor tissue are not known., Methods: The distributions and staining intensities of different molecular forms of type I collagen in ovarian neoplasms were studied immunohistochemically with antibodies to the aminoterminal propeptide of type I procollagen (PINP) and the cross-linked carboxyterminal telopeptide of type I collagen (ICTP), reflecting the presence of newly synthesized and old, cross-linked type I collagen, respectively., Results: A regular pattern of moderately staining, relatively uniform fibers was observed in the stroma of benign serous and mucinous cystadenomas, indicating limited participation in tumor growth. The staining was accentuated subepithelially in borderline epithelial neoplasms and in well differentiated cystadenocarcinomas, suggesting induction of the stromal collagen synthesis by the tumor cells. Fewer degraded collagen fibers were found in moderately differentiated carcinomas, most likely because of enzymatic degradation of the stroma surrounding the neoplasms during tumor spread. Strongly staining, irregular collagen fibers occurred closely around islets of tumor cells in undifferentiated malignant neoplasms and in metastases of ovarian carcinomas; also, intracellular staining was present in part of the malignant cells. In most cases, the staining reactions obtained with the two different antibodies were similar, probably indicating rapid processing of the newly synthesized type I collagen (indicated by PINP) to a maturely cross-linked form (indicated by ICTP)., Conclusions: Synthetic and degradative processes are typical of the collagenous matrix in malignant ovarian tumors. Aberrant expression of type I collagen may occur in anaplastic ovarian carcinomas.

There is a growing body of evidence indicating that prostate-specific antigen (PSA) may be present in many steroid hormone-stimulated epithelial tissues other than that of the prostate. In particular, breast tumor cell lines treated with steroid hormone receptor agonists, breast tumors, and normal human breast have recently been found by our group to contain PSA. To investigate whether PSA may also be present in other human tumors, we employed a highly sensitive immunofluorometric assay technique to quantify PSA immunoreactivity in tumor extracts. Using a PSA-positivity cutoff value of 0.005 ng per mg of protein, 23 of 43 diverse tumors tested positive for PSA protein. Confirmatory analyses for PSA by a commercially available method (IMx) on six samples demonstrated a high degree of concordance between the two methods. To establish the molecular weight of the immunoreactive species, the most highly positive tumor extracts of each tumor type were fractionated by high performance liquid chromatography. Whereas the majority of tumors had immunoreactivity eluting at both 100 KDa and 33 KDa, corresponding to PSA bound to alpha 1-antichymotrypsin and free PSA, respectively, the colon and parotid tumors displayed immunoreactivity only at the 33 KDa fraction. We conclude that in addition to breast tumors and normal breast, colon, ovarian, liver, kidney, adrenal, and parotid tumors can also produce PSA. The physiological role of PSA in these tumors is currently under investigation.

We made an effort to identify a reliable source for obtaining large quantities of both free (PSA) and PSA-ACT complex for the preparation of the calibrator for the PSA assay. Using size exclusion chromatography, we found both free PSA and PSA-ACT complex in the conditioned cell medium of the LNCaP cell line, which was derived from a human metastatic adenocarcinoma of the prostate. An assay specific for PSA-ACT reacted only with the PSA-ACT complex from cells grown in serum-free medium, and not with the complex from the cell medium grown in 10% calf serum. We also found both free PSA and PSA-ACT complex in 15% of cytosols prepared from breast tumor tissues; the cytosol PSA concentrations ranged from 0.1 to 110 ng/ml. No correlation was found between cytosol PSA and concentrations of estrogen receptor, progestin receptor, epidermal growth factor receptor, cathepsin D, or the ectodomain of c-erbB-2 protein. Based on chromatographic characterizations and the slope of their dose-response curves, it appears that both free PSA and PSA-ACT complex found in the cytosols are similar to PSA complex from the cell medium and the serum of prostate cancer patients. Ectopic PSA was also detected in pooled sera from patients with breast, ovarian, pancreatic, and colon carcinoma. The PSA concentrations in these serum pools increased with the level of their dominant tumor marker. In any event, the LNCaP cell medium appears to be a reliable source for obtaining both free and ACT-complexed PSA of human tumor origin for the preparation of PSA assay calibrators.

Objective: We examined the specific expression of gelatinase/type-IV collagenase and tissue inhibitor of metalloproteinase (TIMP) in clinical ovarian cancer tissue., Methods: Molecular weight-specific gelatinase/type-IV collagenase activity was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis in which substrate was included (zymography). The expression of TIMP mRNA was examined by Northern blot analysis., Results: Zymography revealed that in ovarian cancer the activity of a 92-kDa gelatinase/type-IV collagenase was always greater than that of a 64-kDa gelatinase/type-IV collagenase in contrast to the situation in the normal ovary. Northern blot analysis revealed no remarkable difference of TIMP mRNA expression between cancer and normal ovarian tissues., Conclusions: These results indicate that the higher activity of the 92-kDa gelatinase/type-IV collagenase enzyme, relative to that of the 64-kDa enzyme, is involved in the malignant phenotype of ovarian cancer, while the inhibitor of these enzymes, TIMP, is distributed in a widespread fashion in the tissue, and its levels are not correlated with the malignancy.

Background: Clinical drug resistance is recognized in patients previously treated with radiotherapy and after chemotherapy. In vitro exposure of mammalian tumor cells to fractionated X-irradiation also resulted in the expression of drug resistance. Analysis of the resistance phenotype of irradiated Chinese hamster ovary sublines revealed P-glycoprotein overexpression, without any concomitant increase in P-glycoprotein messenger RNA, under posttranslational regulation. This study aimed to determine whether this distinctive resistance phenotype could also be identified in irradiated human tumor cells., Methods: Irradiated sublines established from two human ovarian tumor cell lines, SK-OV-3 and JA-T, which showed resistance to vincristine and to etoposide, were studied. Protein and RNA expression were quantitated by Western and Northern blotting or RNase protection assays. P-glycoprotein turnover was measured after immunoprecipitation of metabolically labelled cells., Results: Significant P-glycoprotein overexpression was detected using the C219 and C494 monoclonal antibodies in the two irradiated human ovarian tumor sublines. No concomitant increase in P-glycoprotein messenger RNA was detectable in the SK-OV-3/DXR10 subline, contrasting with the increased message characteristic of vincristine-selected SKVCR sublines. In addition, turnover of P-glycoprotein was significantly reduced in these DXR10 cells when compared with that measured in a vincristine-selected subline. These irradiated sublines showed reduced levels of epidermal growth factor receptors and unchanged levels of topoisomerase II, but they overexpressed c-erbB2 marginally and heat shock protein 27 significantly. These latter elevations in protein levels, however, were associated with concomitant increases in their respective messenger RNAs, implicating regulation at the transcriptional level., Conclusions: Exposure of human ovarian tumor cells to fractionated X-irradiation in vitro resulted in the expression of a distinctive multiple drug resistance phenotype unusually involving posttranslational regulation of P-glycoprotein. Monitoring tumor biopsies for P-glycoprotein-associated drug resistance in patients treated with radiotherapy should evaluate protein levels rather than, or as well as, MDR1 mRNA expression.

Background: Small cell carcinomas (SCC) are the most common ovarian tumors associated with hypercalcemia. Parathyroid hormone-related protein (PTHrp) is the most frequent cause of hypercalcemia of malignancy., Methods: The presence of PTHrp in SCC has been studied by immunohistochemistry in formalin fixed, paraffin embedded tissues. A polyclonal antibody against a synthetic peptide corresponding to the first 36-N terminal amino acid residues of PTHrp was used. Normal dog skin, which is rich in PTHrp, was used as a positive control. Absorption tests using the synthetic peptide as antigen were done in all the cases., Results: Immunoreactivity for PTHrp was found in five of seven cases of SCC. The serum calcium levels were elevated in two of these cases, normal in two, and unknown in one. The two negative cases were associated with high serum calcium levels., Conclusion: The lack of correlation between immunoreactivity and serum calcium levels can be explained on the basis that immunohistochemistry is dependent on the peptide content of cells rather than on the capability for hormone production and/or release. The results suggest that PTHrp plays a role in the development of hypercalcemia in patients with SCC of the ovary.

Background: Interleukin-6 (IL-6) is a multifunctional cytokine that can be produced by human ovarian cancer cells. Elevated IL-6 levels have been found in the serum and ascites of patients with ovarian cancer, but its role in this disease has not been clearly established., Methods: The authors studied the relationship between IL-6 levels in serum and ascites, various tumor parameters, and survival in 70 patients with newly diagnosed, untreated epithelial ovarian cancer. Ascites and serum specimens were obtained at the time of initial surgery, and IL-6 levels were determined using the B9 bioassay., Results: All patients underwent platinum-based chemotherapy after initial surgery. The median age of the group was 62 years (range, 28-87 years), and the median follow-up time was 13 months (range, 12-59 months). Significantly higher IL-6 levels were detected in patients' ascites (median, 49,612 pg/ml [range, < 1 to 680,330 pg/ml]) compared with serum (median, 10 pg/ml [range, < 1 to 1221 pg/ml]) (P < 0.0001). IL-6 levels in ascites correlated significantly with the volume of ascites (P < 0.0001) and nearly so with the size of tumor found at initial surgery (P = 0.05). Serum and ascites IL-6 levels did not correlate statistically with overall survival time, tumor stage, grade, histologic findings, residual tumor volume after debulking, and serum CA 125 levels. Although not statistically significant, patients who responded to chemotherapy tended to have lower ascites IL-6 levels (median, 21,102 pg/ml) compared with patients who did not respond to chemotherapy (median, 40,200 pg/ml)., Conclusions: IL-6 is present in very high amounts in the ascites of patients with epithelial ovarian cancer. IL-6 levels in ascites correlated significantly with ascites volume and initial tumor size. IL-6 levels in ascites and serum did not correlate statistically with other tumor parameters or with survival time.

Background: Recent studies have shown that multiple cytokines are secreted by ovarian epithelial cancer cells. Previous studies have shown that the cancer cell lines secrete macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 (IL-1), interleukin-6 (IL-6), and transforming growth factor-alpha (TGF-alpha). Concomitantly, the serum levels of one of the growth factors (M-CSF) was found to be significantly elevated in patients with primary ovarian cancer and in second-look patients. The authors evaluated the serum levels of IL-1 alpha, IL-1 beta, IL-6, and tumor necrosis factor-alpha (TNF-alpha) in patients with primary ovarian epithelial cancer. These levels were then compared with cytokine concentration found in normal peritoneal fluid., Methods: Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of cytokines in normal peritoneal fluid, ascites, and serum., Results: In serum, TNF-alpha and IL-6 were significantly increased in primary ovarian cancer patients when compared with control subjects (P < 0.0001 for both cytokines). TNF-alpha and IL-6 were also significantly higher than the levels found in second-look patients (P < 0.007 for TNF-alpha, and P = 0.0002 for IL-6). The levels of IL-1 alpha and beta were not elevated in ovarian cancer. TNF-alpha in the ascites was higher when compared with normal peritoneal fluid and was statistically significantly different when a cut-off point between 71-110 pg was selected (P < 0.005). The levels of IL-6 in ascites from patients with primary ovarian cancer also showed a marked increase (P < 0.0001) when compared with peritoneal fluid from control subjects., Conclusions: Levels of IL-1, IL-6, and TNF-alpha were determined in normal peritoneal fluid, ovarian malignant ascites, normal serum, and serum from patients with ovarian cancer. This study showed that the patients with ovarian cancer have elevated levels of IL-6 and TNF-alpha in serum and ascitic fluid. A larger study would help in evaluating the potential use of cytokines as tumor markers in ovarian cancer.

Background: Tetranectin (TN), a tetrameric, plasminogen-binding protein, was reduced in the plasma of patients with cancer and appears extracellularly in "stimulated" connective tissues, such as the proliferative, connective tissue response to carcinomas known as desmoplasia., Methods: Tissue samples from 37 patients with ovarian cancer were examined immunohistochemically for stromal and cellular TN. Plasma samples obtained before the primary surgery were quantitated for TN. The univariate log-rank test and the multivariate Cox proportional hazards regression model were used to analyze the prognostic function of the variables., Results: A significantly higher survival rate was found for patients with a low-stromal TN score and a high-plasma TN concentration, whereas the cellular TN score did not have any significance. A significant negative correlation was found between plasma TN and stromal TN (RS = -0.36; P = 0.03). Independent significant correlations were found between stromal immunoreactivity for TN and tumor grade (R = 0.67; P = 0.03) and between plasma TN and tumor stage (R = -0.29; P = 0.01)., Conclusions: This study gives great expectations to TN as a useful parameter for prognostic evaluation of patients with ovarian cancer. According to the correlations, stromal TN may partly originate from plasma and enhance proteolytic degradation in the interstitial tissue, a process necessary for the spread and growth of cancer. Because plasma TN measurements are only valid when taken preoperatively, it is of great value that stromal TN immunoreactivity of stored tumor tissue can be used as a prognostic factor for ovarian cancer.

Background: Haptoglobin is an acute-phase reactant protein involved in immune regulation. It has been isolated from malignant ovarian ascites and has been shown to be capable of mediating nonspecific immune suppression in vitro in humans and in vivo in experimental animals. The range of concentrations under which such a nonspecific suppression is observed lies well within concentrations of haptoglobin observed in vivo. This immune suppression could adversely affect patient outcome., Methods: Based on this information, ascites haptoglobin levels of 21 consecutive patients with ovarian cancer who underwent initial surgical debulking were determined. After the patients received chemotherapy, they were divided into those having a positive and those have a negative second-look operation., Results: Seventeen patients with positive pathology at second look had an initial mean ascites haptoglobin level of 99 +/- 49 mg/dl. The mean haptoglobin level in the four patients with negative findings was 67 +/- 42 mg/dl. The difference in these mean haptoglobin levels is not statistically significant (P > 0.05)., Conclusion: Although the number of patients in this study is small, the wide range of values documented in both groups make it doubtful that an initial absolute ascites haptoglobin level will prove clinically prognostic of eventual disease status at the time of second look laparotomy.

Background: Although the ascites of patients with ovarian cancer has been reported to contain immunosuppressive factors, the identity and source of this activity has not been determined. Previously, the authors showed that conditioned media from two of four epithelial ovarian cancer cell lines inhibits proliferation of mitogen-stimulated human lymphocytes. The physical characteristics of the inhibitory substance are unlike those of peptide growth factors but closely resemble those of fibronectin., Methods and Results: In the current study, it was found that the two ovarian cancer cell lines that produce the inhibitory substance have more fibronectin on the cell surface and secrete significantly more immunoreactive fibronectin into their culture media than the other two ovarian cancer cell lines. In addition, the immunosuppressive activity was bound to a gelatin-Sepharose affinity column, known to bind fibronectin. Finally, in ascites from 20 patients with advanced epithelial ovarian cancer, fibronectin levels correlated with the ability to inhibit proliferation of lectin-stimulated lymphocytes (P < 0.001)., Conclusions: Fibronectin is produced by some ovarian cancer cell lines and acts to inhibit proliferation of mitogen-stimulated lymphocytes. Additional studies are needed to clarify the role of fibronectin in ovarian cancer.

The clinicopathologic features of 44 serous borderline tumors (SBT) of the ovary were evaluated. Nineteen were Stages II and III, and 9 had invasive peritoneal implants. All 19 patients received chemotherapy and 4, who had invasive implants, died of disease after 3, 4.3, 8, and 9 years. The other 25 patients were free of tumor 1-14 years (mean, 5.3 years) after surgery. Coexpression of low molecular weight keratins (AE1, CAM 5.2) and vimentin was found in all tumors and their implants. No significant differences were found between SBT with different volume-corrected mitotic indices (M/Vi) with respect to gross features, presence or absence of implants, stage, and survival. Cytometric DNA analysis also was performed on the primary ovarian tumors and the implants. Twenty-one primary tumors had diploid or tetraploid histograms, and 23 had aneuploid histograms. DNA ploidy of the primary ovarian tumors did not correlate with gross features, the presence or absence of implants, M/Vi, stage, and survival. The data from this study confirm that most SBT are clinically benign, but SBT with invasive implants may behave aggressively. Expression of intermediate filaments, M/Vi, and DNA ploidy evaluation of the primary ovarian tumors seem to be of no value in predicting prognosis. However, four of seven patients with aneuploid DNA implants died of tumor.

Eighteen carcinomas involving both the endometrium and the ovary were studied. Stage, size, bilaterality and pattern of ovarian involvement, histologic types and grades, presence of endometrial hyperplasia or ovarian endometriosis, myometrial, tubal, lymphatic and blood vessel invasion, and follow-up of the patients were all evaluated. Accordingly, the cases were classified as follows: Group A (nine cases), two separate primary tumors; and Group B (nine cases), uterine primaries with ovarian metastasis or ovarian primaries with uterine metastasis. Immunohistochemical stains (CAM 5.2, wide-spectrum keratin, vimentin, carcinoembryonic antigen (CEA), CA 12.5, CA 19.9) were performed in all cases. Flow cytometric determination of nuclear DNA was done in 13 (seven Group A and six Group B tumors). Of the nine cases with independent primary tumors, seven showed different immunohistochemical profiles in the ovarian and uterine tumors, whereas only four of the nine metastatic ones had similar staining characteristics. Five cases with independent primary tumors, but only one of the metastatic group, exhibited different aneuploid stemlines in the endometrial and ovarian tumors. The other seven (two independent and five metastatic) cases had similar DNA indexes in both tumors. Immunohistochemical and DNA flow cytometric study may be of some value for the distinction between metastatic and independent tumors, but differential diagnosis must presently rely largely upon conventional clinicopathologic criteria.

The activity of sequentially administered hormonal therapy was investigated over 25 days in 25 patients with epithelial ovarian carcinoma who had estrogen receptor (ERc)-positive tumors. Patients received ethinyl estradiol (EE) (50 micrograms/d) on days 1 to 7 and medroxyprogesterone acetate (MPA) (400 mg/d) on days 8 to 25. Twenty-three patients completed one or more courses of treatment. There were no complete responses (CR). Four partial responses (PR) with durations of 9, 4, 3, and 1 months were seen. Two incomplete responses with durations of 6 and 4 months were also seen. Six patients had stable disease (SD), and 11 patients had progression. The overall response rate was 17% and may represent a modest improvement in response over those in previously published studies conducted with MPA alone. No significant toxic effects were noticed, and some patients reported an improved sense of well-being. However, two patients experienced depression with this treatment. The mean ERc values in responders, patients with SD, and nonresponders were 70.0, 36.7, and 47.9 fmol/mg cytosolic protein, respectively. Future studies of hormonal therapy in patients with ovarian carcinoma should attempt to identify more reliable indices for determining sensitivity to these agents.