Ovarian Cancer Ascites Research Articles

Ascites do not affect the rate of complete cytoreductive surgery and prognosis in patients with primary ovarian cancer with ascites treated with hyperthermic intraperitoneal chemotherapy.

Cytoreductive surgery (CRS) is the current standard therapy procedure for patients with advanced ovarian cancer (OC), but numerous patients with OC are complicated with ascites. The aim of the present study was to assess whether massive ascites affect the rate of complete CRS and prognosis for patients with primary OC treated with hyperthermic intraperitoneal chemotherapy (HIPEC). Between December 2006 and December 2015, 1,293 patients with primary OC from the Intracelom Hyperthermic Perfusion Therapy Center of the Cancer Hospital of Guangzhou Medical University prospective database were treated with CRS combined with HIPEC. A total of 1,225 patients were without malignant ascites or small amounts of ascites and 68 had massive malignant ascites. The rate of complete CRS, overall survival (OS), disease-free survival (DFS) and resolution of ascites for patients with massive ascites were analyzed between patients without/small ascites, and with massive ascites. Complete CRS was successful in 86.8% (1,063/1,225) of patients without/small ascites, and 85.3% (58/68) of patients with massive ascites. No statistical differences were identified in complete CRS success between patients with ascites and patients without/small ascites (P=0.080). For patients with massive ascites, all symptoms exhibited regression; the total objective remission rate was 100% (68/68), even for patients with incomplete CRS (10/68) (P=0.100). The mean OS was 58 months and the mean DFS was 26 months in patients without/small ascite, vs. 57 months and 28 months in patients with massive ascites. No significant differences were noted in median DFS and median OS between patients with ascites, and patients without/small ascites (All P>0.05). In conclusion, the results of the present study suggest that ascites does not affect the rate of complete CRS and the prognosis of patients with massive ascites following HIPEC. CRS is suitable for the majority of patients with primary OC and massive ascites.

A novel TNFR2 antibody induces T cell co-stimulation and promotes durable anti-tumor immunity

Abstract TNFR2, an emerging therapeutic target in immuno-oncology, is upregulated upon T cell activation and is expressed by tumor-infiltrating effector and regulatory T (Treg) cells. We generated a novel monoclonal antibody, Y9, specific to murine TNFR2 and investigated its mechanism of action. In vitro, Y9 stimulation of purified T cells increased proliferation and effector function, indicating that Y9 acts as an agonist and can provide co-stimulation. In vivo, Y9 treatment of mice with established tumors resulted in complete tumor clearance across a variety of models. The activity of Y9 on immune cells was confirmed by its decreased activity in mice depleted of NK or CD8+ T cells. Unlike the proposed Treg depletion mechanism of other therapeutic antibodies, depletion of Treg cells is not the primary mechanism of action of Y9 treatment. Instead, decreased TNFR2 and other co-inhibitory receptor surface expression was observed following treatment. Y9 activity depended on FcγR binding, which facilitated enhanced agonist activity. Based on this evidence, we developed MM-401, a human antibody targeting TNFR2. MM-401 has agonistic activity; upon incubation of MM-401 with human CD4+ and CD8+ T cells, we observed upregulation of activation markers and cytokine production comparable to MEDI6469 (anti-OX40). We also observed that MM-401 promotes antibody-dependent cellular cytotoxicity (ADCC) in an NK cell-mediated in vitro assay and a reduction in the number of Treg cells in human ovarian cancer ascites. These data suggest that MM-401 could also promote anti-tumor immunity by mediating ADCC, as well as by direct co-stimulation of T cell responses, and justifies the continued development of MM-401 as a novel cancer immunotherapy.

Ascites from ovarian cancer patients stimulates MUC16 mucin expression and secretion in human peritoneal mesothelial cells through an Akt-dependent pathway

BackgroundCA125 is a well-established ovarian cancer (OC) serum biomarker. The CA125 heavily glycosylated epitope is carried by the MUC16 mucin, a high molecular weight transmembrane mucin. Upon proteolytic cleavage, the extracellular domain of MUC16 is released from the cell surface into malignant ascites and blood vessels. Previous studies have shown that both tumor and surrounding mesothelial cells may express MUC16. Although little is known about the regulation of MUC16 expression in these cells, recent evidence suggest that inflammatory cytokines may stimulate MUC16 expression. Because malignant ascites is a pro-inflammatory environment, we investigated whether OC ascites stimulate the expression and release of MUC16 by human peritoneal mesothelial cells (HPMCs).MethodsHPMCs were isolated from peritoneal lavages of women operated for conditions other than cancer. MUC16 protein expression was determined by immunoblot, immunofluorescence or immunohistochemistry depending on the experiments. The release of MUC16 from the cell surface was measured using EIA and MUC16 mRNA expression by ddPCR.ResultsWe show that high-grade serous ascites from patients with OC (n = 5) enhance MUC16 expression in HPMCs. Malignant ascites, but not benign peritoneal fluids, stimulate the release of MUC16 in HPMCs in a dose-dependent manner, which is abrogated by heat inactivation. Moreover, we establish that ascites-induced MUC16 expression occurs at the post-transcriptional level and demonstrate that ascites-induced MUC16 expression is mediated, at least partially, through an Akt-dependent pathway. A cytokine array identified upregulation of several cytokines and chemokines in ascites that mediate MUC16 upregulation versus those that do not, including CCL7, CCL8, CCL16, CCL20, CXCL1, IL-6, IL-10, HGF and IL-1 R4. However, when individually tested, none of these factors affected MUC16 expression or secretion. Concentrations of CA125 in the serum of a given patient did not correlate with the ability of its corresponding ascites to stimulate MUC16 release in HPMCs.ConclusionsCollectively, these data indicate that mesothelial cells are an important source of MUC16 in the context of ovarian cancer and malignant ascites is a strong modulator of MUC16 expression in HPMCs and uncover the Akt pathway as a driving factor for upregulation of MUC16. Factors in ascites associated with enhanced MUC16 expression and release remains to be identified.

A Novel ALDH1A1 Inhibitor Targets Cells with Stem Cell Characteristics in Ovarian Cancer.

A small of population of slow cycling and chemo-resistant cells referred to as cancer stem cells (CSC) have been implicated in cancer recurrence. There is emerging interest in developing targeted therapeutics to eradicate CSCs. Aldehyde-dehydrogenase (ALDH) activity was shown to be a functional marker of CSCs in ovarian cancer (OC). ALDH activity is increased in cells grown as spheres versus monolayer cultures under differentiating conditions and in OC cells after treatment with platinum. Here, we describe the activity of CM37, a newly identified small molecule with inhibitory activity against ALDH1A1, in OC models enriched in CSCs. Treatment with CM37 reduced OC cells’ proliferation as spheroids under low attachment growth conditions and the expression of stemness-associated markers (OCT4 and SOX2) in ALDH+ cells fluorescence-activated cell sorting (FACS)-sorted from cell lines and malignant OC ascites. Likewise, siRNA-mediated ALDH1A1 knockdown reduced OC cells’ proliferation as spheres, expression of stemness markers, and delayed tumor initiation capacity in vivo. Treatment with CM37 promoted DNA damage in OC cells, as evidenced by induction of γH2AX. This corresponded to increased expression of genes involved in DNA damage response, such as NEIL3, as measured in ALDH+ cells treated with CM37 or in cells where ALDH1A1 was knocked down. By inhibiting ALDH1A1, CM37 augmented intracellular ROS accumulation, which in turn led to increased DNA damage and reduced OC cell viability. Cumulatively, our findings demonstrate that a novel ALDH1A1 small molecule inhibitor is active in OC models enriched in CSCs. Further optimization of this new class of small molecules could provide a novel strategy for targeting treatment-resistant OC.

PREDICTORS OF CHEMORESISTANCE TO PLATINUM-CONTAINING NEOADJUVANT CHEMOTHERAPY IN ADVANCED OVARIAN CANCER

Chemoresistance is one of the main causes of treatment failure in advanced ovarian cancer (OC). In order to identify predictors of chemoresistance, 30 patients with ascitic form of OC, who received neoadjuvant chemotherapy (NACT) according to AP regimen, were examined. In the epithelial cells of ascites and in tumor tissue after NACT, expression of the VEGFa, ABCB1 and ERCC1 genes was assessed by PCR-RT. The effectiveness of NACT was assessed using the criteria of therapeutic pathomorphosis, survival - using the Kaplan-Meier criterion, the relationship of clinical and molecular parameters - using the Mann-Whitney criterion. Results. We established that in 43% of patients the response to NACT was absent (CRS1), the significant response (CRS2) was in 35% of cases, and the complete response (CRS3) was in 21% of cases. VEGFa expression was increased in ascites in 17% of cases, in tumor tissue - in 33% in the CRS1 group. In groups with CRS 2,3, overexpression of VEGFa in ascites was detected in 25% of cases, in tumor tissue in 37.5%. ABCB1 mRNA expression was increased after NACT in 50% of cases in the CRS1 group and in 25% of cases in the CRS 2,3 group. Thus, platinum-based NACT initiates an increase in the expression of VEGFa and ABCB1 in tumor tissue. We established that in case of high expression (ERCC + / VEGF + / ABCB1 +) in ascites, there is a statistically significant increase in relapse-free survival compared with the group with low or absent expression (p = 0.012). The overall survival rate was 41.8 months in patients with overexpression of the studied genes in the tissue of the primary tumor after NACT and 25.3 months in patients with low expression (p = 0.058). Conclusion. The levels of ERCC1, VEGFa and ABCB1 mRNA in ascitic cells before NACT and in the primary tumor after NACT according to the AP scheme can serve as a predictor of the effectiveness of this treatment in ascitic OC.

Ascitic ovarian cancer is an adequate preclinical model of carcinomatosis to study intraperitoneal chemoperfusion treatment

To study the mechanisms underlying the effects of intraperitoneal chemoperfusion and to develop the optimal chemoperfusion regimen, an animal model of peritoneal carcinomatosis closely resembles a human model of peritoneal carcinomatosis is required. In our study, the model of peritoneal carcinomatosis in rats with ascitic ovarian cancer was used. material and methods. There were three groups of rats with ascitic ovarian cancer: 1 – the control group (n=15) having no treatment; 2 – rats receiving normothermic intraperitoneal chemoperfusion with cisplatin, 40 mg/kg (n=12); 3 – rats receiving hyperthermic intraperitoneal chemoperfusion with cisplatin, 20 mg/kg (n=14). All animals were euthanized with subsequent autopsy. results. Ascitic ovarian cancer developed in 100 % of the animals injected with the tumor cells. The median overall survival of rats in the control group was 9.5 days. At autopsy, all rats had ascites, and rats surviving 15‒17 days after the tumor cell injection had white tumor nodes measuring 1–3 mm in the greater omentum, intestinal mesentery, parietal and visceral peritoneum. The nodes were histologically verified as metastases of low-differentiated ovarian tumor. In 2 and 5 rats from groups 2 and 3 respectively, metastases in paratracheal lymph nodes and tumor hydrothorax were detected with no evidence of peritoneal carcinomatosis. The median survival of rats in groups 2 and 3 was significantly higher than that in the control group, being 37.5 and 25.5 months, respectively (р=0,256). conclusion. This in vivo study proved that localization of ascitic ovarian tumor, development of the tumor in all animals injected with tumor cells, fast ascites progression and peritoneal carcinomatosis make this ascitic ovarian cancer an adequate preclinical model of peritoneal carcinomatosis to study intraperitoneal chemoperfusion. Further studies are needed to understand the reasons and mechanisms of the tumor hydrothorax development in rats after intraperitoneal chemoperfusion.

Ascites as a subject of studies in ovarian cancer

Ovarian cancer is a highly heterogeneous disease characterized by multiple histological subtypes. High- grade serous ovarian carcinoma is the most common histological subtype of ovarian cancer. The majority of ovarian cancer patients present with malignant ascites at diagnosis. Peritoneal dissemination is one of the most unfavorable factors for tumor progression and recurrence. A more precise visualization of peritoneal carcinomatosis can be achieved by transabdominal ultrasound. However, the prognostic factors associated with malignant ascites in ovarian cancer are currently not well understood. Among the clinical parameters, the volume of ascites has the greatest information in terms of prognosis of disseminated ovarian cancer. Ovarian cancer with small-volume ascites has a more favorable therapeutic prognosis. Ascites is an easily accessible and valuable source of cellular and extracellular components contained in it that are involved in ovarian carcinogenesis. Ascites represents an accessible and valuable source of material to identify signals that influence tumor growth. At present, among the soluble high- and low molecular components of ascites, an active search for additional prognostic and predictor factors is being conducted, providing insights into the molecular mechanisms for clinical phenotypes of ovarian cancer.

Usefulness of cell-free and concentrated ascites reinfusion therapy in the therapeutic management of advanced ovarian cancer patients with massive ascites.

The management of refractory ascites in advanced ovarian cancer (AOC) is vital for patients with abdominal distention, respiratory distress, and anorexia due to massive ascites with cancer peritonitis. We analyzed the benefits of concentrated ascites reinfusion therapy (CART) in the management of AOC. We reviewed records of AOC patients who underwent CART between January 2011 and March 2017. We retrospectively analyzed patients' backgrounds and physiological changes, including body weight, abdominal girth, urine volume, blood component values, blood pressure, heart rate, and body temperature before and after CART. We investigated the clinicopathological significance of CART by measuring the mean number of ascites tumor cell (ATC) clusters before CART. A retrospective analysis was performed on 29 cases of AOC with massive ascites involving 47 CART sessions. The patients' mean age was 56.6 ± 12.8 years, and the mean number of sessions was 1.7 ± 1.2. The mean volume of the processed ascites was 2,937 ± 820mL, which was concentrated to 272 ± 84mL containing 85.0 ± 33.2g protein on average. Significant reductions in abdominal girth (- 5.30 ± 0.65cm; p < 0.0001) and body weight (- 2.97 ± 0.26kg; p = 0.0011), increased urine volume (+ 824.29 ± 145.21mL; p < 0.0001), and improved serum albumin levels (+ 0.18 ± 0.34; p < 0.0001) were observed after CART. Analysis of variance revealed significant elevations in body temperature after CART in 11 patients with a small number of ATC clusters. CART is useful for the therapeutic management of AOC patients with refractory massive ascites. Elevations of body temperature after CART may be avoided by the investigation of patients' peritoneal cytology before CART.

Effective Targeting of TAG72+ Peritoneal Ovarian Tumors via Regional Delivery of CAR-Engineered T Cells.

Impressive clinical efficacy of chimeric antigen receptor (CAR)-engineered T cell therapy for hematological malignancies have prompted significant efforts in achieving similar responses in solid tumors. The lack of truly restricted and uniform expression of tumor-associated antigens, as well as limited T cell persistence and/or tumor trafficking pose major challenges for successful translation of CAR T cell therapy in solid tumors. Recent studies have demonstrated that aberrantly glycosylated cell surface proteins on tumor cells are amenable CAR targets. Tumor-associated glycoprotein 72 (TAG72) antigen is the sialyl-Tn found on multiple O-glycoproteins expressed at high levels on the surface of several cancer types, including ovarian cancer. Here, we developed a humanized TAG72-specific CAR containing a 4-1BB intracellular co-stimulatory signaling domain (TAG72-BBζ). TAG72-BBζ CAR T cells showed potent antigen-dependent cytotoxicity and cytokine production against multiple TAG72+ ovarian cancer cell lines and patient-derived ovarian cancer ascites. Using in vivo xenograft models of peritoneal ovarian tumors, regional intraperitoneal delivery of TAG72-BBζ CAR T cells significantly reduced tumor growth, extended overall survival of mice, and was further improved with repeat infusions of CAR T cells. However, reduced TAG72 expression was observed in early recurring tumors, which coincided with a lack of T cell persistence. Taken together, we demonstrate efficacy with TAG72-CAR T cells in ovarian cancer, warranting further investigations as a CAR T cell therapeutic strategy for this disease.

Novel therapeutic strategies for advanced ovarian cancer by using induced pluripotent stem cell-derived myelomonocytic cells producing interferon beta.

Although first‐line chemotherapy has a high rate of complete responses in ovarian cancer patients, the vast majority of patients present with recurrent disease that has become refractory to conventional chemotherapy. Peritoneal dissemination and malignant ascites are the hallmarks of recurrent or advanced ovarian cancer and severely reduce quality of life. Development of therapeutic measures to treat such patients is eagerly anticipated. Macrophage infiltration is observed in various types of cancer including epithelial ovarian cancer. In addition, macrophages are involved in the formation of spheroids in the malignant ascites of ovarian cancer and promote cancer growth. iPS‐ML, macrophage‐like myelomonocytic cells generated from human induced pluripotent stem (iPS) cells, made close contacts with ovarian cancer cells in vitro. We hypothesized that, if we inoculate iPS‐ML‐producing IFN‐β (iPS‐ML/IFN‐β) into the peritoneal cavity of patients with ovarian cancer, IFN‐β produced by the iPS‐ML/IFN‐β would efficiently act on the cancer cells to suppress cancer growth. To evaluate this hypothesis, we injected iPS‐ML/IFN‐β into SCID mice bearing peritoneally disseminated human ovarian cancer cells, SKOV3. Immunohistochemical analysis of the intraperitoneal tumors detected iPS‐ML/IFN‐β infiltrating into the cancer tissues. Therapy with iPS‐ML/IFN‐β significantly suppressed tumor progression. In addition, dramatic reduction of cancer‐related ascites was observed. Collectively, it is suggested that iPS‐ML/IFN‐β therapy offers a new approach for the treatment of patients with advanced ovarian cancer.

DAXX promotes ovarian cancer ascites cell proliferation and migration by activating the ERK signaling pathway

BackgroundThe death-domain-associated protein (DAXX) was originally identified as a protein that binds to the transmembrane death receptor FAS and enhances both FAS-induced and transforming growth factor-β-dependent apoptosis. In a previous study, we found that nude mice injected with DAXX-overexpressing cells (ES-2-DAXX) accumulated large concentrations of first-generation ascites cells (I ascites cells). The role of DAXX in the development of ascites is unknown. The aim of this study was to analyze the effect of DAXX on proliferation and migration of ascites cells in ovarian cancer in vitro and in vivo.MethodsNude mice were housed in cages with a 14:10 h light:dark cycle; water and food were provided ad libitum. ES-2-DAXX cells (1×106) were injected intraperitoneally into athymic nude mice (8-week-old female mice). After 4 weeks, I ascites cells were collected. The I ascites cells were injected intraperitoneally into athymic nude mice (8-week-old female mice). After 4 weeks, II ascites cells were collected and cultured. Ascites cell survival, migration, and colony formation were measured using colony formation and cell growth assays. Immunofluorescent staining revealed the co-localization of DAXX and promyelocytic leukemia protein (PML) in ascites cell nuclei. Western blotting and immunohistochemistry showed that extracellular signal-related kinase (p-ERK) 1/2 and CEBP-β were highly expressed in tumor tissues formed by II ascites cells. Through immunoprecipitation, we also found that DAXX can interact with CEBP-β.ResultsDAXX enhanced ascites cell survival, migration, and colony formation. DAXX and PML nuclear foci dramatically increased in a passage-dependent manner in ascites cells, DAXX promoted the tumor growth of ascites cells in vivo, increased ascites cell proliferation in vivo, and enhanced ascites cell survival and migration by activating the ERK signalling pathway and integrating with CEBP-β.ConclusionsDAXX can interact with CEBP-β. DAXX can induce ovarian cancer ascites formation by activating the ERK signal pathway and binding to CEBP-β.

977P - The role of bevacizumab plus front-line chemotherapy in patients with malignant ascites of ovarian cancer

977P - The role of bevacizumab plus front-line chemotherapy in patients with malignant ascites of ovarian cancer

21P - Cisplatin in NIPEC or HIPEC?

Background: Eliminating minimal residual disease in patients with advanced ovarian cancer (AOC) is necessary to prevent recurrencies and could be achieved with hyperthermic intraperitoneal chemoperfusion (HIPEC). Cisplatin comprises the majority of HIPEC regimens due to its known synergic efficacy with hyperthermia. However, emerging report are impugning its role. Our goal was to compare the survival of rats treated with cisplatin by hyperthermic and normothermic intraperitoneal chemoperfusion (HIPEC and NIPEC) and to evaluate, whether the outcome of HIPEC would depend on its opened (oHIPEC) or closed (cHIPEC) delivery. Methods: A rodent model of ascitic ovarian cancer was used. 1*107 tumor cells were inoculated i.p. 48 hours prior to the treatment to 48 female Wistar rats. As indicated by our previous research, this time limit of tumor progression reflects the biological pattern of optimally debulked AOC in women. Each 12 rats were randomized into four groups to receive either cisplatin at a maximum tolerated dose of 20 mg/kg in NIPEC, cHIPEC, and oHIPEC or i.p. 20 mg/kg saline as a control without treatment. Results: The mean survival in the untreated control was 31.8 days. Cisplatin in NIPEC increased the mean survival by 22 days (P = 0.0007), while in cHIPEC and oHIPEC by 19.9 (P = 0.003) and 31.5 (P = 0.003) days, respectively. Cisplatin in oHIPEC was shown to be the most effective combination. The differences between NIPEC and cHIPEC with cisplatin were not statistically significant. Conclusions: Our results prompt that NIPEC with cisplatin might be just as effective as cHIPEC in increasing survival in AOC. Legal entity responsible for the study: Vladimir Bespalov. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.

Abstract A23: Neuropilin-1 expression on regulatory T cells in ovarian cancer

Abstract Introduction: Regulatory T cells (Treg) are a subpopulation of CD4+ T cells that suppress autoimmune responses, but also prevent clearance of tumors and chronic viral infections. In ovarian cancer, a higher frequency of tumor-infiltrating Treg is associated with poor prognosis, but insights into the mechanisms governing the survival and suppressive activity of Treg in this context are limited. Our group has recently described a signaling axis through neurophilin-1 (NRP1) on Treg in murine models of cancer that promotes the survival and suppressive function of Treg. Genetic knockout of NRP1 on murine Treg leads to reduced tumor growth and increased survival, underscoring the importance of NRP1+ Treg in suppressing antitumor immunity. Given the importance of NRP1+ Treg in murine cancer models, we sought to explore the expression pattern of NRP1 on tumor-infiltrating Treg from patients with ovarian cancer. Materials and Methods: Ovarian tumors were obtained through the health sciences tissue bank at the University of Pittsburgh, and ovarian ascites fluid was obtained through Magee Women’s Research Institute. Peripheral blood was obtained from healthy donors through the Red Cross. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation, and tumor-infiltrating lymphocytes were isolated by first mechanically disrupting the tumor, followed by treatment for 15 minutes at 37°C with 50 ug/mL Liberase DL. For flow cytometric analysis of Treg, cells from blood and tumors were first stained for surface markers, followed by live/dead discrimination, fixation and permeabilization, and staining for intracellular markers. Cells were analyzed on an LSR Fortessa at the Hillman Cancer Center Flow Core. For functional assays, Treg were selected from ascites fluid and assayed for their ability to suppressive naïve CD8+ T cells ex vivo. Wilcoxon rank sum tests were used to assess differences between groups, and a two-sided alpha less than 5% was considered significant. Results: A total of 12 healthy donors and 20 ovarian cancer patients were studied. Intracellular and surface NRP1 expression was assessed by flow cytometry on CD4+CD25+FOXP3+ Treg from healthy donor peripheral blood and tumor-infiltrating lymphocytes from either ovarian cancer tumors or ascites fluid from patients with ovarian cancer. Intracellular NRP1 was expressed on a median of 1.8% (interquartile range [IQR]: 0.69% to 4.8%) of Treg from healthy donors compared with a median of 66% (IQR: 28% to 90%; p&amp;lt;0.001) and 82% (IQR: 38% to 99%; p=0.004) of Treg from ovarian tumor-infiltrating lymphocytes (TIL) and ascites, respectively. Surface NRP1 was detected on 0.1% (IQR: 0% to 1%) of Treg in PBMC from healthy donors, compared with 11.1% (IQR: 1.1% to 60%; p=0.01) on ovarian TIL and 82% (IQR: 14% to 82%; p=0.0015) on ascites. Comparing expression of intracellular NRP1 on Treg from N=2 benign ovarian tissues versus N=14 malignant ovarian tumors, there was a trend towards increased NRP1 expression (median 9.3% versus 54.5%, respectively; p=0.044). Finally, Treg isolated from ascites fluid were capable of suppressing CD8+ T cells ex vivo proliferation in a dose-dependent manner. Conclusions and Future Directions: Both intracellular and surface NRP1 are expressed more frequently on tumor-infiltrating Treg and Treg from ascites fluid compared to Treg from healthy donor peripheral blood. Although the number of patients currently included in this study is small, there was also evidence that intracellular NRP1 expression on Treg was higher on malignant compared with benign tissue. Additionally, Treg from ascites fluid were capable of suppressing CD8 cells ex vivo. Future studies will evaluate whether blockade of NRP1 on human Treg limits either the survival or suppressive capacity of Treg. Note: This abstract was not presented at the conference. Citation Format: Anthony R. Cillo, Tullia Bruno, Francesmary Modugno, Robert Edwards, Dario Vignali. Neuropilin-1 expression on regulatory T cells in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A23.

Ascites IL-10 Promotes Ovarian Cancer Cell Migration

Ovarian cancer (OC) ascites is an inflammatory and immunosuppressive tumor environment characterized by the presence of various cytokines, chemokines and growth factors. The presence of high concentrations of these cytokines/chemokines in ascites is associated with a more aggressive tumor phenotype. IL-10 is an immunosuppressive cytokine for which high expression has been associated with poor prognosis in some cancers. However, its role on OC tumor cells has not been explored. Therefore, the aim of the current study was to elucidate the role of ascites IL-10 on the proliferation, migration and survival of OC cell lines. Here, we show that IL-10 levels are markedly increased in patients with advanced serous OC ascites relative to serous stage I/II ascites and peritoneal effusions from women with benign conditions. Ascites and IL-10 dose-dependently enhanced the proliferation and migration of OC cell lines CaOV3 and OVCAR3 but had no effect on cell survival. IL-10 levels in ascites positively correlated with the ability of ascites to promote cell migration but not proliferation. Depletion of IL-10 from ascites markedly inhibited ascites-induced OC cell migration but was not crucial for ascites-mediated cell proliferation. Taken together, our findings establish an important role for IL-10, as a component of ascites, in the migration of tumor cells.

Abstract 5318: High-grade serous ovarian cancer DNA methylation and survival in African-American women

Abstract Ovarian cancer is the most lethal gynecologic cancer in the United States, and African-American (AA) women have the poorest outcomes compared to other racial/ethnic groups. Although several biomarkers have been proposed to establish prognosis in European ancestry (EA) patients, including some DNA methylation markers (FGF4, FGF21, MYLK2, MYLK3, MYL7, and ITGAE) (Phelps et al., 2017), it is unknown if these or other markers are applicable to AA patients. Using data from the African-American Cancer Epidemiology Study (AACES), we evaluated 1) if DNA methylation is associated with residual disease and survival, and 2) if previously reported CpG biomarkers for EA women are related to survival in AA women. 121 AA women with high-grade serous ovarian cancer (HGSOC) were randomly selected from 600 women enrolled in AACES. Clinical records and formalin-fixed, paraffin-embedded (FFPE) tumor tissue were retrieved, and a pathologist reviewed histopathologic slides to confirm diagnosis. 92 HGSOC had complete clinical information. DNA methylation was measured using Illlumina HumanMethylationEPIC. Beta-values were preprocessed (RELIC, Tost+BMIQ and ComBat). Low quality probes were filtered. Tumor purity was estimated using InfiniumPurify; &amp;lt;30% was considered low purity. Four cell estimates (RefFreecellmix) were used as a proxy of cell composition. To evaluate DNA methylation alterations, we used the top 100K most variable CpG sites and a semisupervised recursively partitioned mixture model (ssRPMM) approach to delineate the patients into RPMM classes. We also evaluated the six candidate CpGs from Phelps et al. The dataset was divided into training and validation subsets (50% each); if findings were consistent, a pooled statistic is reported. We used logistic regression to evaluate the association between DNA methylation and residual disease; Cox proportional hazard models were used for survival. Models were adjusted for age at diagnosis, low purity, cell types, neoadjuvant therapy, tissue source (adnexa vs. peritoneum), histology (serous vs. mixed), and residual disease. The RPMM classes were not associated with residual disease. For survival, four RPMM classes were delineated, which we collapsed into two classes. A lower risk of mortality was observed for one of the RPMM classes, HR: 0. 03[95% CI: 0.01-0.12]. This “low risk” RPMM class grouped five CpGs in genes PLEC1, AP5B1, DNAH7 and MAPK15. These genes have been associated with cell motility and ovarian cancer ascites. Among the candidate CpGs, we only observed a trend to better survival per every 10% increase in MYLK3 CpG methylation, HR: 0.51 [95%CI: 0.24-1.10]. These preliminary results suggest that some DNA methylation modifications may identify subgroups of AA women with better survival. Previously reported biomarkers in EA may not be as useful in AA women. Future studies with increased sample size may help to disentangle these associations. Citation Format: Lucas A. Salas, Lauren C. Peres, Sarah E. Abbott, Casey S. Greene, Jeffrey R. Marks, Anthony J. Alberg, Elisa V. Bandera, Jill S. Barnholtz-Sloan, Ann G. Schwartz, Michele L. Cote, Patricia G. Moorman, Ellen M. Funkhouser, Edward S. Peters, Melissa L. Bondy, Paul D. Terry, Jennifer A. Doherty, Brock C. Christensen, Joellen M. Schildkraut. High-grade serous ovarian cancer DNA methylation and survival in African-American women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5318.

Single-cell analysis of EMT-related gene expression heterogeneity in metastatic tumor cell clusters isolated from ovarian cancer ascites

Single-cell analysis of EMT-related gene expression heterogeneity in metastatic tumor cell clusters isolated from ovarian cancer ascites

Vitamin D-Binding Protein Enhances Epithelial Ovarian Cancer Progression by Regulating the Insulin-like Growth Factor-1/Akt Pathway and Vitamin D Receptor Transcription.

Purpose: Malignant ascites of epithelial ovarian cancer (EOC) helps identify prognostic biomarkers or mechanisms of tumor progression. Vitamin D-binding protein (DBP) was revealed to be upregulated in EOC ascites in our previous proteomic study. Here, we examined the role of DBP in EOC.Experimental Design: We analyzed ascites, serum, and tissue samples of patients with newly diagnosed EOC to determine the prognostic effects of DBP. We verified DBP function using orthotopic animal models and DBP regulation in ovarian cancer cell lines.Results: Elevated ascitic DBP was significantly associated with poor response to chemotherapy, short progression-free interval, increased cancer progression, and death. Ascitic DBP overexpression was an independent unfavorable biomarker for progression-free survival; DBP overexpression in cancerous tissue was significantly related to chemoresistance. In vivo and in vitro investigations demonstrated an important role for DBP in ovarian cancer progression. Orthotopic model mice inoculated with DBP knockdown ovarian cancer cells displayed a significant reduction in tumor formation, malignant cell number, ascitic DBP levels, invasiveness, and metastasis, and increased survival compared with controls. In presence of vitamin D receptor (VDR), DBP promoted cell aggression (invasion and doubling time) via activation of the insulin-like growth factor-1/insulin-like growth factor-binding protein-2/Akt axis, and induced suppression of vitamin D-responsive genes. A NF-κB p65-binding site in the VDR promoter was identified as a major determinant of DBP-dependent VDR promoter activation.Conclusions: This study highlights the importance of DBP in ovarian tumor progression and the potential application of DBP as a therapeutic target for EOC. Clin Cancer Res; 24(13); 3217-28. ©2018 AACR.

HE4 level in ascites may assess the ovarian cancer chemotherapeutic effect

BackgroundThe clinical treatment of ovarian cancer with ascites is problematic. The main reasons for treatment failure are the susceptibility to intraperitoneal metastasis and chemotherapeutic drug resistance. The purpose and significance of this study is to evaluate which marker might evaluate treatment efficacy and improve the cure rate.ResultsThis study compared a no chemotherapy group with a chemotherapy group regarding the determination of carbohydrate antigen 125 and human epididymis protein 4 in ovarian cancer ascitic supernatants and cross-analyzed routine serum carbohydrate antigen 125 levels. The level of human epididymis protein 4 in the ascites of the chemotherapy group was significantly lower than that of the no chemotherapy group (p < 0.001). Moreover, the expression of ascitic human epididymis protein 4 correlated positively with serum carbohydrate antigen 125 levels (p < 0.001). MDR was positive in 13 of the 30 samples (43.33%) in the chemotherapy group with highly expressed CA125.ConclusionThe level of human epididymis protein 4 in ovarian cancer ascites may reflect the therapeutic effect of ovarian cancer patients, and a high level of human epididymis protein 4 might predict chemoresistance and the possibility of ascites formation. The determination of the expression of human epididymis protein 4 alone or combined with carbohydrate antigen 125 levels in both serum and ascites in ovarian cancer patients with ascites may have important significance for guiding and improving the treatment regimen.

Hypocrellin B-loaded, folate-conjugated polymeric micelle for intraperitoneal targeting of ovarian cancer invitro and invivo.

Photodynamic therapy (PDT) is considered an innovative and attractive modality to treat ovarian cancer. In the present study, a biodegradable polymer poly (ethylene glycol) (PEG)‐poly (lactic acid)(PLA)‐folate (FA‐PEG‐PLA) was prepared in order to synthesize an active‐targeting, water‐soluble and pharmacomodulated photosensitizer nanocarrier. Drug‐loading content, encapsulation efficiency, in vitro and in vivo release were characterized, in which hypocrellin B (HB)/FA‐PEG‐PLA micelles had a high encapsulation efficiency and much slower control release for drugs compared to free drugs (P < .05). To evaluate the targeting ability of the HB/FA‐PEG‐PLA micelles, a cellular uptake study in vitro was carried out, which showed significantly enhanced uptake of HB/FA‐PEG‐PLA micelles in SKOV3 (FR+) compared to A2780 cancer cells (FR−). The enhanced uptake of HB/FA‐PEG‐PLA micelles to cancer cells resulted in a more effective post‐PDT killing of SKOV3 cells compared to plain micelles and free drugs. Binding and uptake of HB/FA‐PEG‐PLA micelles by SKOV3 cells were also observed in vivo after ip injection of folate‐targeted micelles in tumor‐bearing ascitic ovarian cancer animals. Drug levels in ascitic tumor tissues were increased 20‐fold (P < .001), which underscored the effect of a regional therapy approach with folate targeting. Furthermore, the HB‐loaded micelles were mainly distributed in kidney and liver (the main clearance organs) in biodistribution. These results showed that our newly developed PDT photosensitizer HB/FA‐PEG‐PLA micelles have a high drug‐loading capacity, good biocompatibility, controlled drug release, and enhanced targeting and antitumor effect, which is a potential approach to future targeting ovarian cancer therapy.