Search

Abstract: Objectives: Current antigen-based diagnostic assays for ovarian cancers rely on intravasation of specific aberrantly expressed proteins and their achieving detectable steady-state concentrations, resulting in their inability to truly detect small early lesions. In contrast, tumor antigen immunorecognition is observed following initial transformation events. Our objective was to characterize humoral antitumor responses in terms of IgG subclasses generated and tumor antigens recognized. Methods: For patients with benign and malignant ovarian disease, tumor-reactive IgG subclasses were characterized by Western immunoblotting. Antigen recognition patterns were analyzed by 2-dimensional electrophoresis and proteins exhibiting shared or stage-specific recognition were defined by mass spectrometry (MS) sequencing. Results: Sera from ovarian cancer patients exhibited significantly greater immunoreactivities than either controls or women with benign disease. While late-stage patients recognized more proteins at greater intensity, stage-specific differential recognition patterns were observed in the IgG subclasses, with the greatest recognition appearing in IgG2 subclasses. Immunoreactivity in IgG2 and IgG3 from stage I and II patients appears to be most intense with nuclear antigens >40 kDa, while, in stage III patients, additional immunoreactivity was present in the <40 kDa components. Stage III patients also exhibited similar reaction with membrane antigens <40 kDa. Two-dimensional electrophoresis revealed 32 stage-linked antigenic differences with 11 in early-stage and 21 in late-stage ovarian cancer. Conclusions: Owing to the timing and stability of humoral responses, quantitation of IgG subclasses recognizing specific tumor antigens provides superior biomarkers for early cancer identification and allows for differentiation of benign versus malignant ovarian masses and early- and late-stage cancers. [Copyright &y& Elsevier]

Abstract: Objective: Most ovarian cancers are diagnosed at advanced stage (67%) and prospects for significant improvement in survival reside in early diagnosis. Our objective was to validate our array assay for the identification of ovarian cancer based on quantitation of tumor-reactive IgG. Methods: The diagnostic array utilizes specific exosome-derived antigens to detect reactive IgG in patients'' sera. Specific protein targets were isolated by immunoaffinity from exosomes derived from ovarian tumor cell lines. Sera were obtained from age-matched female volunteers, women with benign ovarian disease and with ovarian cancer. Immunoreactivity was also compared between exosomal proteins and their recombinant counterparts. Results: Sera from ovarian cancer patients exhibited significantly greater immunoreactivities than either normal controls or women with benign disease (both considered negative to all antigens tested). Reactivities with nucleophosmin, cathepsin D, p53, and SSX common antigen for patients with all stages of ovarian cancer were significantly higher than for controls and women with benign ovarian disease. Reactivity with placental type alkaline phosphatase, TAG 72, survivin, NY-ESO-1, GRP78, and Muc16 (CA125) allowed the differentiation between Stage III/IV and early stage ovarian cancer. Conclusions: The quantitation of circulating tumor-reactive IgG can be used to identify the presence of ovarian cancer. The analyses of IgG recognition of specific exosomal antigens allows for the differentiation of women with benign ovarian masses from ovarian cancer, as well as distinguishing early and late stage ovarian cancers. Thus, the quantitative assessment of IgG reactive with specific tumor-derived exosomal proteins can be used as diagnostic markers for ovarian cancer. [Copyright &y& Elsevier]

Abstract: Objectives: Most ovarian cancer patients are diagnosed at an advanced stage (67%) and prospects for significant improvement in survival reside in early diagnosis. While expression patterns of a recently identified biomarker family, microRNA, appear to be characteristic of tumor type and developmental origin, microRNA profiling has been limited to tissue specimens. Tumors actively release exosomes into the peripheral circulation and we now demonstrate the association of microRNAs with circulating tumor-derived exosomes. Methods: Circulating tumor exosomes were isolated using a modified MACS procedure with anti-EpCAM. Initially, microRNA profiles of ovarian tumors were compared to those of tumor exosomes isolated from the same patients. Levels of 8 microRNAs (miR-21, miR-141, miR-200a, miR-200c, miR-200b, miR-203, miR-205 and miR-214) previously demonstrated as diagnostic, were compared in exosomes isolated from sera specimens of women with benign disease and various stages of ovarian cancer. Results: MicroRNA from ovarian tumor cells and exosomes from the same patients were positive for 218 of 467 mature microRNAs analyzed. The levels of the 8 specific microRNAs were similar between cellular and exosomal microRNAs (exhibiting correlations from 0.71 to 0.90). While EpCAM-positive exosomes were detectable in both patients with benign ovarian disease and ovarian cancer, exosomal microRNA from ovarian cancer patients exhibited similar profiles, which were significantly distinct from profiles observed in benign disease. Exosomal microRNA could not be detected in normal controls. Conclusions: These results suggest that microRNA profiling of circulating tumor exosomes could potentially be used as surrogate diagnostic markers for biopsy profiling, extending its utility to screening asymptomatic populations. [Copyright &y& Elsevier]

Abstract: Treatment failures result from resistance to chemotherapy in ovarian cancer. The effect of cisplatin and paclitaxel treatments on chemosensitivity was studied in ovarian cancer cells developed from a patient with stage IIIC disease. Cells (UL-3A, UL-3B) that recovered from cisplatin (Cis) and paclitaxel (Tax) treatments showed higher levels of p53, mdr-1 and chemoresistance than untreated controls. EC50 values of Cis and Tax for UL-3A clones were 7.2–34.6, average 20.9μg/ml, while UL-3B clones ranged from 11.8–252.0μg/ml, with a mean value of 73.2μg/ml for Cis, and 260.0–4400.0nM (mean 2555.0nM) for Tax. Selection pressures during treatment may contribute to drug resistance. [Copyright &y& Elsevier]

Objective. In women with advanced ovarian cancer, levels of CD3-zeta on peripheral blood lymphocytes have previously been demonstrated to correlate with responsiveness to interleukin (IL)-2 therapy. The aims of this study were to identify the circulating component that modulated zeta expression and to define whether this suppressive activity could serve as a marker of biotherapy responsiveness.Methods. Sera were obtained from 17 patients with advanced ovarian cancer treated with intraperitoneal IL-2 in a phase I trial between 1987 and 1990. Nine of these patients exhibited a clinical response, while eight did not respond. Six additional sera from age-matched, noncancer-bearing women were used as controls. Jurkat E6-1 cells were used to assay modulation of CD3-zeta by sera and serum-derived components. Jurkat cells were exposed to serum or chromatographically fractionated serum components for 4 days and zeta expression was analyzed by Western immunoblots and quantitated by densitometry.Results. The effect of sera on zeta expression was compared between responders and nonresponders. Incubation of Jurkat cells with sera from responders suppressed CD3-zeta expression by 36.7% (vs. control treated), while treatment with sera from nonresponders produced an 83.7% reduction in zeta expression (difference between groups, P < 0.001). When sera from nonresponders were chromatographically fractionated, a <20 kDa component was identified that correlated with decreased zeta chain expression. This component was diminished in the sera of responders and absence in controls.Conclusions. Thus, in patients with advanced ovarian cancer, a circulating component, which decreases zeta expression, can be identified as a marker for responsiveness to IL-2 therapy. [Copyright &y& Elsevier]

Objectives:The presence of CD44 isoforms was evaluated in ascitic fluid and serum samples of patients with gynecologic malignancies. Previously, the shedding of tumor-associated cell surface antigens has been demonstrated in the blood and malignant effusions of gynecologic cancer patients. Thus, the shedding of CD44 was also studied in ascitic fluids and sera of these patients, to address variant isoform expression as a biomarker of gynecologic cancer.Methods:The expression of CD44 isoforms by ovarian tumor cells was examined by flow cytometry using variant-specific monoclonal antibodies. The release of these isoforms into the peripheral circulation and ascites was assayed by Western immunoblot analysis.Results:Flow cytometric analysis of ovarian tumor cell lines revealed a strong expression of CD44 with significant levels of v4/5 and v6 isoforms. The presence of circulating CD44 isoforms was detectable in the sera of six of eight cancer patients, as well as in 12 of 16 ascitic fluids. Of the CD44-positive specimens, all six positive sera expressed detectable levels of variant CD44. The CD44v6 was present in all of the positive sera samples tested. In the ascites, the “shed” CD44 appeared to be associated predominantly with shed particles (vesicles) of plasma membranes (membrane fragments). Of ten CD44-positive ascites samples, all expressed significant levels of variant CD44.Conclusions:In addition to mediating metastasis, the differential expression and shedding of CD44 isoforms into the circulation may represent important deteminants in the escape of tumors from immune surveillance, and their detection may be a diagnostic or prognostic marker. [ABSTRACT FROM PUBLISHER]

Copyright of Obstetrics & Gynecology is the property of Lippincott Williams & Wilkins and Its content may not be copied or emailed to multiple sites or posted to a listserv without the. [Extracted from the article]

Abstract: Cell-derived vesicles are recognized as essential components of intercellular communication, and many disease processes are associated with their aberrant composition and release. Circulating tumor-derived vesicles have major potential as biomarkers; however, the diagnostic use of exosomes is limited by the technology available for their objective characterization and measurement. In this study, we compare nanoparticle tracking analysis (NTA) with submicron particle analysis (SPA), dynamic light scattering (DLS), and electron microscopy (EM) to objectively define size distribution, number, and phenotype of circulating cell-derived vesicles from ovarian cancer patients. Using the NanoSight LM10 instrument, cell-derived vesicles were visualized by laser light scattering and analyzing Brownian motion of these vesicles captured by video. The NTA software calculates the size and total concentration of the vesicles in solution. Using vesicles isolated from ovarian cancer patients, we demonstrate that NTA can measure the size distributions of cell-derived vesicles comparable to other analysis instrumentation. Size determinations by NTA, SPA, and DLS were more objective and complete than that obtained with the commonly used EM approach. NTA can also define the total vesicle concentration. Furthermore, the use of fluorescent-labeled antibodies against specific markers with NTA allows the determination of the “phenotype” of the cell-derived vesicles. [Copyright &y& Elsevier]