Ovarian Cancer Ascites Research Articles

The FACIT-AI, a new tool for assessing symptoms associated with malignant ascites

ObjectiveThe objectives of this study are to assess the clinical relevance and validity of the Functional Assessment of Chronic Illness Therapy-Ascites Index (FACIT-AI) in women with ovarian cancer and malignant ascites, and to modify the instrument guided by qualitative feedback from patients with recurrent malignant ascites. MethodsFourteen adult female patients with recurrent symptomatic malignant ascites were enrolled from three centers. All completed an open-ended symptom list to identify their primary concerns regarding their condition. They then completed a draft 10-item FACIT-AI questionnaire created from expert input. Eleven patients provided comments regarding the FACIT-AI questionnaire using a written feedback format. Three patients participated in a “think-aloud” cognitive debriefing interview to ensure patient comprehension of questionnaire items. ResultsOf the first 11 patients surveyed, 7 believed that the draft FACIT-AI contained all important symptoms associated with malignant ascites. Responses from the remaining 4 patients revealed three symptoms that 2 or more patients nominated for inclusion: urinary frequency, constipation and emotional distress. These items were added to the original FACIT-AI to produce a 13-item index of symptoms associated with malignant ascites. ConclusionsThe 13-item FACIT-AI has content validity among women with malignant ascites associated with ovarian cancer. It is available for use in clinical research or practice, with the expectation that more will be learned about its performance and interpretation over time.

Osteoprotegerin (OPG) protects ovarian cancer cells from TRAIL-induced apoptosis but does not contribute to malignant ascites-mediated attenuation of TRAIL-induced apoptosis

BackgroundResistance to apoptosis is a major problem in ovarian cancer and correlates with poor prognosis. Osteoprotegerin (OPG) is a secreted factor in malignant ascites and acts as a decoy receptor for receptor activator of NF-κB ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL promotes apoptosis in ovarian cancer cells. Ovarian cancer ascites attenuate TRAIL-induced apoptosis raising the possibility that OPG contained in ascites may abrogate the anti-tumor activity of TRAIL.MethodsDetermination of OPG levels in ascites was measured by ELISA. Effect of OPG on TRAIL-induced cell death was determined by XTT and colony forming assays in ovarian cancer cell lines and primary tumor cells. Apoptosis was assessed by ELISA.ResultsWe found that recombinant OPG and malignant ascites attenuates TRAIL-induced cell death and apoptosis in a dose-dependent manner in ovarian cancer cell lines and primary ovarian tumor cells. OPG is present at high levels in the ascites of patients with ovarian cancer. We found a positive correlation between the levels of OPG in ascites and the ability of the ascites to attenuate TRAIL-induced cell death. The anti-apoptotic effect of ascites was not reversed by co-incubation with an OPG blocking antibody.ConclusionsOPG and malignant ascites protect ovarian cancer cells from TRAIL-induced apoptosis. Although malignant ascites contain high levels of OPG, OPG is not a critical component that contributes to ascites-mediated attenuation of TRAIL-induced apoptosis.

Ovarian cancer stem cells: Working towards the root of stemness

Despite medical advances made over the past decade, ovarian cancer remains one of the more lethal gynecologic cancers in the United States. While current therapeutic strategies are relatively effective, there is a high incidence of recurrent chemoresistant disease. This has been attributed, in part, to a regenerative tumor cell sub-population that has acquired stem cell properties which allows these cells to escape standard chemotherapeutics and drive recurrent disease. To date, a number of laboratories have identified these cancer stem cell (CSC) sub-populations in ovarian cancer cell lines, tumors or ascites and the collective findings suggest ovarian CSCs are likely to be as heterogeneous as the disease itself. Moreover, the multiple ovarian histophenotypes and possible sites of disease origin together with the potential for differential hierarchal contributions of multiple CSCs populations represent significant challenges to the identification, functional characterization and therapeutic targeting of ovarian CSC. This review will highlight the markers and methodology currently used to identify and isolate these cells. We will discuss some of the underlying ovarian CSC biology, the signaling pathways implicated in their survival, replication and differentiation and potential therapeutic targeting strategies.

New zinc(II), palladium(II) and platinum(II) complexes of dl-piperidine-2-carboxylic acid; X-ray crystal structure of trans-[Zn2(μ-Ca)2(Hpa)2Cl6] and anticancer activity of some complexes

New complexes of dl-piperidine-2-carboxylic acid (dl-H2pa), [Zn(Hpa)(AcO)(H2O)2], trans-[Zn2(μ-Ca)2(Hpa)2Cl6], [M(bpy)(Hpa)]Cl and [M(pa)(PPh3)2] (M(II)=Pd, Pt) have been prepared and characterized on the basis of elemental analyses, molar conductivity and thermal measurements, IR, Raman, UV–Vis, NMR (1H and 31P) and mass spectroscopy. dl-Piperidine-2-carboxylic acids act as bidentate ligands, through the carboxyl oxygen and cyclic nitrogen atoms. The crystal structure of trans-[Zn2(μ-Ca)2(Hpa)2Cl6], obtained from the addition of ZnCl2 to dl-H2pa in either hard tap water or presence of CaCl2, has been determined by X-ray diffraction. It crystallizes in a triclinic lattice with space group symmetry P1. The complex has two zinc atoms in tetrahedral geometry, each ligated by a carboxyl oxygen and three chlorine atoms. The other carboxyl oxygen atoms from the two Hpa− ligands are bridged by two calcium atoms, i.e., there are two Zn(Hpa−)Cl3 units bridged by two calcium atoms. The free dl-H2pa and its complexes, trans-[Zn2(μ-Ca)2(Hpa)2Cl6], [Pd(bpy)(Hpa)]Cl and [M(pa)(PPh3)2] (M(II)=Pd, Pt) have been tested against the serous ovarian cancer ascites, OV 90 cell line.

Protein markers of cancer-associated fibroblasts and tumor-initiating cells reveal subpopulations in freshly isolated ovarian cancer ascites

BackgroundIn ovarian cancer, massive intraperitoneal dissemination is due to exfoliated tumor cells in ascites. Tumor-initiating cells (TICs or cancer stem cells) and cells showing epithelial-mesenchymal-transition (EMT) are particularly implicated. Spontaneous spherical cell aggregates are sometimes observed, but although similar to those formed by TICs in vitro, their significance is unclear.MethodsCells freshly isolated from malignant ascites were separated into sphere samples (S-type samples, n=9) and monolayer-forming single-cell suspensions (M-type, n=18). Using western blot, these were then compared for expression of protein markers of EMT, TIC, and of cancer-associated fibroblasts (CAFs).ResultsS-type cells differed significantly from M-type by expressing high levels of E-cadherin and no or little vimentin, integrin-β3 or stem cell transcription factor Oct-4A. By contrast, M-type samples were enriched for CD44, Oct-4A and for CAF markers. Independently of M- and S-type, there was a strong correlation between TIC markers Nanog and EpCAM. The CAF marker α-SMA correlated with clinical stage IV. This is the first report on CAF markers in malignant ascites and on SUMOylation of Oct-4A in ovarian cancer.ConclusionsIn addition to demonstrating potentially high levels of TICs in ascites, the results suggest that the S-type population is the less tumorigenic one. Nanoghigh/EpCAMhigh samples represent a TIC subset which may be either M- or S-type, and which is separate from the CD44high/Oct-4Ahigh subset observed only in M-type samples. This demonstrates a heterogeneity in TIC populations in vivo which has practical implications for TIC isolation based on cell sorting. The biological heterogeneity will need to be addressed in future therapeutical strategies.

Methods for Identification of CA125 from Ovarian Cancer Ascites by High Resolution Mass Spectrometry

CA125 is the most widely used tumour marker in ovarian cancer with unsatisfactory sensitivity and specificity especially at early stage. It is quantified by antibody-based immunoassays; however different molecular weight isoforms have been described in the literature which have never been validated by mass spectrometry, potentially affecting the diagnostic accuracy and clinical reliability of the test. In this study, CA125 was detected by Western blot and its identity confirmed by mass spectrometry. Two-dimensional (2D) gel electrophoresis in combination with mass spectrometry revealed that positive Western blot signals up to 500 kDa are most likely false-positive interactions of M11-like and OC125-like antibodies. Fibronectin, identified as one of these false-positive interaction partners, increased the reading for CA125 in a first generation ELISA significantly (p = 0.02). The existence of low-molecular weight isoforms of CA125 is therefore questionable and is most likely reflecting cross-reactivity of the antibodies with other proteins. This would explain the conflicting reports on the molecular structure of CA125 and also the inconsistency of CA125 levels by different ELISAs. Our results are also the first steps towards a mass spectrometric assay for CA125 quantification, which would improve sensitivity and reliability.

Elevated and secreted phospholipase A₂ activities as new potential therapeutic targets in human epithelial ovarian cancer.

Ascites in epithelial ovarian cancer (EOC) promotes tumor development by mechanisms that are incompletely understood. Lysophosphatidic acid (LPA), a major tumor-promoting factor in EOC ascites, is an enzymatic product of autotaxin (ATX) and phospholipase A(2) (PLA(2))enzymes. The contribution of PLA(2) activities to ovarian tumorigenesis was investigated. The quantitative measurement of PLA(2) activities in ascites and tissues, as well as assay conditions selective for PLA(2) subtypes, were optimized and validated. PLA(2) activities correlated with tumor-promoting activates in cell-based and in vivo assays. High activities consistent with both cytosolic and calcium-independent PLA(2) were found in human EOC ascites for the first time. Elevated PLA(2) and ATX activities were also observed in EOC compared to benign tumors and normal tissues. Cell-free and vesicle-free (S4) human EOC ascites potently promoted proliferation, migration, and invasion of human EOC cells in a PLA(2)-dependent manner. LPA mediated a significant part of the cell-stimulating effects of ascites. S4 ascites stimulated tumorigenesis/metastasis in vivo, and methyl arachidonyl fluorophosphonate was highly effective in inhibiting EOC metastasis in mouse xenograft models. PLA(2) activity was found in conditioned media from both EOC cells and macrophages. Collectively, our work implies that PLA(2) activity is a potential marker and therapeutic target in EOC.

Interleukin-6 signaling regulates anchorage-independent growth, proliferation, adhesion and invasion in human ovarian cancer cells

It has been widely reported that Interleukin-6 (IL-6) is overexpressed in the serum and ascites of ovarian cancer (OVCA) patients, and elevated IL-6 level correlates with poor prognosis and survival. However, the exact role that IL-6 plays in this malignancy or whether IL-6 can regulate tumorigenic properties has not been established. Here we demonstrate that overexpression of IL-6 in non-IL-6-expressing A2780 cells (by transfecting with plasmid encoding for sense IL-6) increases anchorage-independent growth, proliferation, adhesion and invasion, while depletion of endogenous IL-6 expression in IL-6-overexpressing SKOV-3 cells (by transfecting with plasmid encoding for antisense IL-6) decreases. Further investigation indicates that IL-6 promotes OVCA cell proliferation by altering cell cycle distribution rather than inhibiting apoptosis and that IL-6-enhanced OVCA cell invasive may be associated with increased matrix metalloproteinase (MMP)-9 but not MMP-2 proteolytic activity. In addition, overexpressing or deleting of IL-6 in OVCA cells enhances or reduces its receptor (IL-6Rα and gp130) expression and basal phosphorylation levels of both ERK and Akt, and additional treatment with specific inhibitor of the ERK or Akt signaling pathway significantly inhibits the proliferation of IL-6-overexpressing A2780 cells. Our data suggest that the autocrine production of IL-6 by OVCA cells regulates tumorigenic properties of these cells by inducing IL-6 signaling pathways. Thus, regulation of IL-6 expression or its related signaling pathway may be a promising strategy for controlling the progression of OVCA.

Abstract 1406: A new ovarian cancer metastasis model using multicellular spheroids generated from human ovarian cancers tissues

Abstract Objective Most research aimed at dissecting the mechanism by which epithelial ovarian cancer (EOC) metastasizes has focused on the ability of individual cells to attach and create tumor implants. However, multicellular aggregates known as spheroids are frequently found in ovarian cancer ascites and these aggregates are increasingly thought to play an important role in metastasis not only for ovarian cancer but also other solid tumors where similar phenomena have been observed. We investigated characteristics of spheroids compared to cells grown in monolayer to further characterize these spheroids. Methods IRB approval was obtained for collection of human tissue. Fresh specimens of ovarian cancer were minced, enzymatically digested, rinsed and incubated either in DMEM to generate monolayer cultures or Mammosphere media to generate spheroids. Tumorigenicity was assessed by inoculating individual spheroids or an equal number of dissociated cells into the peritoneal cavity of SCID mice. Gene expression was examined using Western blot and quantitative real-time PCR. Results We found that cells from most ovarian cancers do generate spheroids and most of the spheroids generated are able to reliably induce peritoneal implants in SCID mice. At a molecular level, the spheroids were enriched for expression of specific markers including CD133, CD44, NANOG and OCT4, associated with populations of cancer initiating cells (CICs) as well as other stem cell markers. MiR-26b, characteristic of CICs, was significantly up regulated in spheroids compared to monolayer cells. Spheroids from CD133 positive cells induced ovarian and abdominal tumors in SCID mice, however, CD44 spheroids only generated localized tumor, suggesting that CD133 is important in metastasis of ovarian cancer. Conclusion Our results indicate that populations of cells from human EOC are capable of spontaneously generating multicellular aggregates that resemble the spheroids found in ovarian cancer ascites de novo. These spheroids are enriched for expression of markers including CD133, CD44, NANOG and OCT4, suggesting that spheroid formation enhances stem cell-like markers. CD133 might also serve as a necessary marker for metastasis of ovarian cancer. The increased expression of miR-26b in spheroids compared to monolayer culture is a novel finding that needs further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1406. doi:1538-7445.AM2012-1406

Abstract 4275: PLA2 activities in human epithelial ovarian cancer ascites

Abstract Ascites production is characteristic of late stage human epithelial ovarian cancer (EOC) and it correlates with tumor spread clinically. However, the role of this microenvironment in cancer cells and the mechanisms by which ascites promotes tumor development are not well understood. In recent years, phospholipase A2 (PLA2) enzymes have been identified as cancer therapeutic targets focusing mainly on secreted PLA2s (sPLA2s). In this work, we optimized and validated the quantitative analytic methods to measure PLA2 activities in human EOC ascites and tissues. We found, for the first time, that both cytosolic PLA2 (cPLA2) and calcium-independent PLA2 (iPLA2), but not sPLA2 and autotaxin (ATX), activities (not their expression levels) in human EOC tissues and ascites (the tumor microenvironment) were significantly elevated. In addition, while cell-free (S1) and vesicle-free (S4) human EOC ascites had potent promoting activities in proliferation, migration, and invasion of human EOC cells, the PLA2 activities were involved and account for a significant portion of these activities. Moreover, in vivo studies demonstrated that methyl arachidonyl fluorophosphonate (MAFP, a dual inhibitor of cPLA2 and iPLA2), was highly effective in inhibiting EOC tumorigenesis/metastasis in xenograft models, supporting PLA2 activities as new targets for EOC. LPA, an enzymatic product of ATX and PLA2 enzymes, mediates a significant part of the cellular effects of ascites as evidenced by 1) LPA is produced in cell-free ascites; 2) LPA production and their sensitivities to inhibitors correlate well with the cellular effects in ascites; and 3) the cellular activities were sensitive to LPAR inhibitor and siRNA against LPARs. While cPLA2 and iPLA2 are cytosolic enzymes, our findings indicate that they are associated with cell- and microvesicle-free ascites. In summary, our work implies the potential marker and therapeutic values of PLA2 activity in ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4275. doi:1538-7445.AM2012-4275

O-Linked glycome and proteome of high-molecular-mass proteins in human ovarian cancer ascites: Identification of sulfation, disialic acid and O-linked fucose

The O-linked glycosylation of the main acidic high-molecular-weight glycoprotein from ascites fluid from patients with ovarian cancer were analyzed. The O-linked oligosaccharides were shown to consist of mainly highly sialylated core 1 and 2 structures with a smaller amount of sulfated core 2 structures. These structures were shown to be able to be further extended into small keratan sulfate (KS)-type oligosaccharides with up to four N-acetyllactosamine units. Proteomic studies of the acidic fraction of ascites fluid from patients with ovarian cancer showed that this fraction was enriched in proteoglycans. Among them, lumican, agrin, versican and dystroglycans were potential candidates, with threonine- and serine-rich domains that could carry a significant amount of O-linked glycosylation, including also the O-linked KS. Glycomic analysis using liquid chromatography (LC)-tandem mass spectrometry (MS/MS) also showed that the disialic acid NeuAc-NeuAc- was frequently found as the terminating structure on the O-linked core 1 and 2 oligosaccharides from one ascites sample. Also, a small amount of the epidermal growth factor (EGF)-associated O-linked fucose structure Gal-GlcNAc-Fucitol was detected with and without sialic acid in the LC-MS/MS analysis. Candidate proteins containing O-linked fucose were suggested to be proteoglycan-type molecules containing the O-linked fucose EGF consensus domain.

Separation of kallikrein 6 glycoprotein subpopulations in biological fluids by anion‐exchange chromatography coupled to ELISA and identification by mass spectrometry

Kallikrein 6 (KLK6) has been shown to be aberrantly glycosylated in ovarian cancer. Here, we report a novel HPLC anion exchange method, coupled to a KLK6-specific ELISA, capable of differentiating KLK6 glycoform subgroups in biological fluids. Biological fluids were fractionated using anion exchange and resulting fractions were analyzed for KLK6 content by ELISA producing a four-peak elution profile. Using this assay, the KLK6 elution profile and distribution across peaks of a set (n = 7) of ovarian cancer patient matched serum and ascites fluid samples was found to be different than the profile of serum and cerebrospinal fluid (CSF) of normal individuals (n = 7). Glycosylation patterns of recombinant KLK6 (rKLK6) were characterized using tandem mass spectrometry (MS/MS), and found to consist of a highly heterogeneous KLK6 population. This protein was found to contain all of the four diagnostic KLK6 peaks present in the previously assayed biological fluids. The rKLK6 glycoform composition of each peak was assessed by lectin affinity and MS/MS based glycopeptide quantification by product ion monitoring. The combined results showed an increase in terminal alpha 2-6 linked sialic acid in the N-glycans found on KLK6 from ovarian cancer serum and ascites, as opposed to CSF and serum of normal individuals.

Intravenous aflibercept for treatment of recurrent symptomatic malignant ascites in patients with advanced ovarian cancer: a phase 2, randomised, double-blind, placebo-controlled study

Targeting of VEGF is a potential therapeutic option in patients with malignant ovarian ascites. We present the final results of a multicentre study of the efficacy and safety of aflibercept, a potent inhibitor of both VEGF and placental growth factor, in the treatment of malignant ascites. In this double-blind, placebo-controlled, parallel-group, phase 2 study, patients with advanced chemoresistant ovarian cancer and recurrent symptomatic malignant ascites were randomly assigned (1:1) via an interactive voice response system to either intravenous aflibercept (4 mg/kg every 2 weeks) or placebo, stratified by interval of time (≤ 2 weeks vs > 2 weeks) between the two most recent paracenteses before randomisation. Patients participated in the double-blind period (during which patients, investigators, and sponsor personnel were masked to treatment assignment) until they had a repeat paracentesis and for at least 60 days, and could also participate in an optional open-label period during which all patients received aflibercept. The primary efficacy endpoint was time to repeat paracentesis based on response during the double-blind period alone, and was analysed in the intention-to-treat population with censoring of patients who did not have a repeat paracentesis as of the last day of the double-blind period. Safety analyses included both double-blind and open-label periods. This study is registered at ClinicalTrials.gov, number NCT00327444. 55 patients with a median of four (range two to 11) previous lines of chemotherapy were randomly assigned to receive placebo (n=26) or aflibercept (n=29). Mean time to repeat paracentesis was significantly longer with aflibercept than with placebo (55·1 [SE 7·3] vs 23·3 [7·7] days; difference 31·8 days, 95% CI 10·6-53·1; p=0·0019). In the aflibercept group, two patients did not need a repeat paracentesis during 6 months of double-blind treatment. The most common grade 3 or 4 treatment-emergent adverse events were dyspnoea (six [20%] aflibercept vs two [8%] placebo), fatigue or asthenia (four [13%] vs 11 [44%]), and dehydration (three [10%] vs three [12%]). The frequency of fatal gastrointestinal events was higher with aflibercept (three intestinal perforations) than with placebo (one intestinal fistula leading to sepsis). This study shows the effectiveness of VEGF blockade in the reduction of malignant ascites, but confirms the significant clinical risk of fatal bowel perforation in this population of patients with very advanced cancer. VEGF blockade should be used with caution in advanced ovarian cancer with abdominal carcinomatosis, and the benefit-risk balance should be thoroughly discussed for each patient. Sanofi Oncology.

PGE2-Induced CXCL12 Production and CXCR4 Expression Controls the Accumulation of Human MDSCs in Ovarian Cancer Environment

Signals mediated by CXCL12 (SDF1) and its receptor CXCR4 are centrally involved in cancer progression, both directly by activating cancer cells and indirectly by inducing angiogenesis plus recruiting T regulatory and plasmacytoid dendritic immune cells. Here, we show that in ascites isolated from ovarian cancer patients, both CXCL12 and CXCR4 are controlled by the tumor-associated inflammatory mediator prostaglandin E(2) (PGE(2)), which attracts myeloid-derived suppressor cells (MDSC) into the ascites microenvironment. In this setting, PGE(2) was essential both for expression of functional CXCR4 in cancer-associated MDSCs and for production of its ligand CXCL12. Frequencies of CD11b(+)CD14(+)CD33(+)CXCR4(+) MDSCs closely correlated with CXCL12 and PGE(2) levels in patient ascites. MDSCs migrated toward ovarian cancer ascites in a CXCR4-dependent manner that required COX2 activity and autocrine PGE(2) production. Inhibition of COX2 or the PGE(2) receptors EP2/EP4 in MDSCs suppressed expression of CXCR4 and MDSC responsiveness to CXCL12 or ovarian cancer ascites. Similarly, COX2 inhibition also blocked CXCL12 production in the ovarian cancer environment and its ability to attract MDSCs. Together, our findings elucidate a central role for PGE(2) in MDSC accumulation triggered by the CXCL12-CXCR4 pathway, providing a powerful rationale to target PGE(2) signaling in ovarian cancer therapy.

A phase II study of aflibercept in patients with advanced epithelial ovarian cancer and symptomatic malignant ascites

ObjectiveThe recombinant fusion protein, aflibercept binds and neutralizes vascular endothelial growth factor (VEGF) A, B and placental growth factor (PlGF). Aflibercept inhibits ascites formation and reduces tumor burden in an ovarian cancer model. This open-label, single-arm, multicenter phase II study assessed the efficacy and safety of aflibercept in patients with advanced chemo-resistant epithelial ovarian cancer and symptomatic malignant ascites. MethodsPatients who required ≥3 previous paracenteses at 1-4 paracenteses per month received intravenous aflibercept 4mg/kg every 2weeks. The primary endpoint was repeat paracentesis response rate (RPRR), with response defined as at least a two-fold increase in time to repeat paracentesis compared with the baseline interval. ResultsTen out of 16 enrolled patients achieved a response; the RPRR was 62.5% (95% CI 35.4%–84.8%). Aflibercept was considered effective based on a hypothesis that the RPRR was ≥60%. Median time to repeat paracentesis was 76.0 (95% CI 64.0–178.0) days, which was 4.5 times longer than the baseline interval (16.8days). Median progression-free survival was 59.5 (95% CI 41.0–83.0) days. Twelve patients experienced adverse events considered related to aflibercept treatment including hypertension (7 patients), headache, anorexia, and dysphonia (3 patients each). Two patients experienced Grade 3/4 treatment-related adverse events (Grade 3 hypertension and weight loss in one patient, Grade 3 intestinal perforation in one patient). ConclusionAflibercept 4mg/kg every 2weeks was effective at controlling malignant ascites, reducing the interval between repeat paracenteses. The safety profile was consistent with that reported for anti-VEGF agents.

Prognostic significance of IL-6 and IL-8 ascites levels in ovarian cancer patients

BackgroundThe acellular fraction of epithelial ovarian cancer (EOC) ascites promotes de novo resistance of tumor cells and thus supports the idea that tumor cells may survive in the surrounding protective microenvironment contributing to disease recurrence. Levels of the pro-inflammatory cytokines IL-6 and IL-8 are elevated in EOC ascites suggesting that they could play a role in tumor progression.MethodsWe measured IL-6 and IL-8 levels in the ascites of 39 patients with newly diagnosed EOC. Commercially available enzyme-linked immunosorbent assay (ELISA) was used to determine IL-6 and IL-8 ascites levels. Ascites cytokine levels were correlated with clinicopathological parameters and progression-free survival.ResultsMean ascites levels for IL-6 and IL-8 were 6419 pg/ml (SEM: 1409 pg/ml) and 1408 pg/ml (SEM: 437 pg/ml) respectively. The levels of IL-6 and IL-8 in ascites were significantly lower in patients that have received prior chemotherapy before the surgery (Mann-Whitney U test, P = 0.037 for IL-6 and P = 0.008 for IL-8). Univariate analysis revealed that high IL-6 ascites levels (P = 0.021), serum CA125 levels (P = 0.04) and stage IV (P = 0.009) were significantly correlated with shorter progression-free survival. Including these variables in a multivariate analysis revealed that elevated IL-6 levels (P = 0.033) was an independent predictor of shorter progression-free survival.ConclusionElevated IL-6, but not IL-8, ascites level is an independent predictor of shorter progression-free survival.

Cytokine analysis as a tool to understand tumour–host interaction in ovarian cancer

Epithelial ovarian cancer (EOC) is an immunogenic tumour and exploits many suppressive ways to escape immune eradication. EOC is known to spread primarily by tumour cell implantations in peritoneal cavity. Therefore, ascites may be an ideal fluid compartment to unravel the immune status of the peritoneal cavity.We analysed the expression of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IFN-γ, TNF-α, TNF-β, TGF-β and CCL22 in ovarian cancer ascites, representing immune activating and suppressing cytokines.We observed high expression of pro-inflammatory cytokines IL-6, IL-8 and immune suppressive cytokines IL-10, CCL22 and TGF-β in most samples whereas Th1 (IL-12p70, IFN-γ) and Th2 (IL-4, IL-5) cytokines were only detectable in 13% of the samples. TGF-β was only detected in latent form, questioning its immune suppressive role. CCL22 was in similar levels present in early stage compared to advanced stage tumours. At advanced stage, we observed a negative correlation with CCL22 levels and Th1/2 cytokine expression. We found a positive correlation between IL-6 concentration in ascites and residual disease after debulking. Additionally, IL-6 levels were remarkably higher at recurrence compared to primary advanced disease, which opens an opportunity for inhibition of IL-6 expression in the prevention of recurrence. Despite the heterogeneity of EOC and the complexity of cytokine functions, our results show that cytokine analysis in ascites may aid in understanding tumour–host interaction in EOC.

Abstract 2223: ELISA coupled anion exchange methodology for separation of KLK6 glycoprotein subpopulations in biological fluids

Abstract Ovarian cancer is the leading cause of death among all gynecological disorders. Elevated levels of Kallikrein 6 (KLK6), a secreted trypsin-like N-glycosyalted protease, have been shown to have unfavourable prognostic value in ovarian cancer patients. It has been revealed to be aberrantly up regulated at both transcriptional and translational levels in ovarian cancer. Aberrant glycosylation, or more specifically, increased sialylation of proteins has been observed in the cancer of the ovary. KLK6 isolated from ascites fluid of ovarian cancer patients has been shown to have enrichment of terminal alpha 2-6 linked sialic acid at its single N-glycosylation site when compared to protein isolated from cerebrospinal fluid (CSF), which is believed to be the major source of KLK6 present in circulation. However, due to the relatively low levels of KLK6 in serum (ng/ml range) neither mass spectrometry nor lectin based characterization of this protein found in serum has been achieved. Here, we report a novel HPLC anion exchange method, coupled to a KLK6 specific ELISA, capable of differentiating KLK6 glycoform subgroups in biological fluids, including serum, at physiologically relevant levels. In brief, biological fluids were injected onto a MonoQ bead matrix (GE Healthcare), bound proteins were eluted over a salt gradient and the resulting fractions were analyzed for KLK6 content resulting in a chromatographic elution profile containing four distinct peaks. Utilizing this assay, the KLK6 elution profile and distribution across peaks of a small set (n=8) of ovarian cancer patient matched serum and ascites fluid samples was found to be different than the profile of serum and CSF of normal individuals (n=10). Utilizing tandem mass spectrometry (MS/MS), recombinant KLK6 (rKLK6) purified from an immortalized human cell line was characterized and found to have a highly heterogeneous KLK6 population, encompassing the majority of glycoforms previously shown to be present in the protein from CSF and ovarian cancer ascites. This protein was subjected to the developed assay and was found to contain all of the four diagnostic KLK6 peaks present in the previously assayed biological fluids. Due to the available high quantity of protein from this source (mg levels) we were able to analyze the rKLK6 glycoform composition of each peak utilizing lectin affinity and MS/MS based glycopeptide quantification by single reaction monitoring. The combined results showed a significant increase in terminal alpha 2-6 linked sialic acid on the N-glycans found on KLK6 from ovarian cancer serum and ascites, as opposed to CSF and serum of normal individuals. Therefore, further development and application of this methodology might lead to improvement of KLK6 as a serum ovarian cancer biomarker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2223. doi:10.1158/1538-7445.AM2011-2223

Abstract 4420: Analysis of functional role of interleukin-6 secreted by tumor-associated macrophages in ovarian cancer ascites

Abstract Tumor-associated macrophages (TAMs) are known to contribute to tumor growth, invasion, and metastasis by producing various mediators. However, few studies have clarified the role of TAMs in human ovarian cancer patients. The aim of the present study was therefore to analyze the effect of TAM on ovarian cancer growth, invasion, and metastasis. Ascites were collected aseptically, and CD11b positive cells (TAMs) were purified by positive selection using magnetic-activate cell sorting (MACS) technology. Co-culture of ovarian cancer cell line, SKOV3ip1 cells, with TAMs significantly enhanced the invasive activity of cancer cells in Matrigel Boyden chamber assay. This enhancement was partially blolcked by the treatment of anti-interleukin (IL)-6 receptor antibody (Tocilizumab). Since SKOV3ip1 cells did not express IL-6 in mRNA and protein level, IL-6 secreted by TAMs were considered to play a key role in ovarian cancer invasion. In vivo ovarian cancer xenografts, i.p. treatment of Tocilizumab significantly reduced peritoneal dissemination and ascites formation. Those results suggested targeting IL-6 produced by TAMs could be a promising therapy for a subset of ovarian cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4420. doi:10.1158/1538-7445.AM2011-4420

Abstract 1551: Soluble tissue transglutaminase modulates cellular adhesion to matrix and peritoneal metastasis in ovarian cancer

Abstract Background: Tissue transglutaminase (TG2) is a secreted protein that binds fibronectin (FN) and exerts protein transamidating activity in the presence of Ca2+. We previously reported that TG2 is upregulated in ovarian cancer (OC) cells and enhances intraperitoneal (ip) metastasis. TG2 is secreted abundantly in OC ascites, yet the function of soluble (s) TG2 remains unknown. We hypothesized that sTG2 acts as a permissive factor in the ECM by promoting cancer cell proliferation and adhesion to the matrix. Materials and Methods: Recombinant his6-tagged full length human TG2 and its inactive enzymatic mutant C277S were purified by immobilized metal (Co2+) affinity and anion exchange chromatography. OV90 and A2780 OC cells that do not express endogenous TG2 were used. Solid phase adhesion assay measured adhesion to ECM proteins, MTT assay measured cell proliferation and an ip xenograft model quantified tumor formation and metastasis. Results: S-TG2 enhanced cell adhesion to FN (p = 0.01) and vitronectin (VN, p = 0.01), but not to collagen and laminin. A neutralizing antibody to α5β1 and to αVβ3 blocked sTG2 induced cell adhesion to FN or to VN, respectively, suggesting that sTG2 modulates cell interaction with the matrix via integrins. Digestion of FN with matrix metallo-proteinase 2 blocked OC cell adhesion to digested fragments of FN, while addition of sTG2 restored OC cells’ binding to this protease-digested matrix (p = 0.02). This suggests that the soluble enzyme may facilitate the interaction of OC cells with a protease-remodeled matrix remodeled, similar to the tumor ECM. Enzymatically inactive soluble C277S TG2 enhanced OC cell adhesion to FN to the same extent as wild type TG2. In contrast, an antibody raised against the FN-binding domain of TG2 blocked sTG2 induced OC cell adhesion to FN (p =0.01). These data suggest that the interaction between TG2 and FN, but not the transamidating activity, is important to sTG2-mediated cell interaction with the ECM. When added to conditioned media, sTG2 (2.5ug/mL) increased OC cell proliferation by 20% (p = 0.01). Biweekly ip inoculation of 2.5ug sTG2 increased peritoneal dissemination of OV90 cells in nude mice (11.38 vs. 27.75, p = 0.02), but did not affect tumor volume (412.3 vs. 578.2 mm3, p = 0.34). Conclusions: These data demonstrate that sTG2 increases OC cell adhesion to the ECM and peritoneal metastasis. The key mechanism involved in this process is the interaction between sTG2, FN, and integrins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1551. doi:10.1158/1538-7445.AM2011-1551