Sebastian Szubert

Role of osteopontin in differential diagnosis of ovarian tumors

The aim of this study was to evaluate the role of the serum osteopontin (OPN) level as a biomarker for discriminating between malignant and benign ovarian tumors. Furthermore, comparisons with the diagnostic usefulness of the other tests were performed.

Ovarian cancer-derived ascitic fluids induce a senescence-dependent pro-cancerogenic phenotype in normal peritoneal mesothelial cells

PurposeAfter the seeding ovarian cancer cells into the peritoneal cavity, ascitic fluid creates a microenvironment in which these cells can survive and disseminate. The exact nature of the interactions between malignant ascitic fluids and peritoneal mesothelial cells (HPMCs) in ovarian cancer progression has so far remained elusive. Here we assessed whether malignant ascitic fluids may promote the senescence of HPMCs and, by doing so, enhance the acquisition of their pro-cancerogenic phenotype.MethodsPrimary omentum-derived HPMCs, ovarian cancer-derived cell lines (A2780, OVCAR-3, SKOV-3), malignant ascitic fluids and benign ascitic fluids from non-cancerous patients were used in this study. Ovarian cancer cell proliferation, as well as HPMC proliferation and senescence, were determined using flow cytometry and β-galactosidase assays, respectively. Ovarian cancer cell migration was quantified using a Transwell assay. The concentrations of soluble agents in ascitic fluids, conditioned media and cell lysates were measured using DuoSet® Immunoassay Development kits.ResultsWe found that HPMCs, when exposed to malignant ascitic fluids, exhibited decreased proliferation and increased senescence rates. The malignant ascitic fluids were found to contain elevated levels of HGF, TGF-β1 and GRO-1, of which HGF and GRO-1 were able to induce senescence in HPMCs. We also found that HPMCs subjected to malignant ascitic fluids or exogenously added HGF and GRO-1 stimulated ovarian cancer cell progression, which was manifested by an increased production of HA (adhesion), uPA (proliferation), IL-8 and MCP-1 (migration).ConclusionOur results indicate that malignant ascitic fluids may contribute to ovarian cancer progression by accelerating the senescence of HPMCs.

External validation of the IOTA ADNEX model performed by two independent gynecologic centers

OBJECTIVES The external, two-center validation of the IOTA ADNEX model for differential diagnosis of adnexal tumors. METHODS A total of 204 patients with adnexal masses (134 benign and 70 malignant) treated at the Division of Gynecologic Surgery, Poznan University of Medical Sciences, Poland (Center I), and 123 patients (89 benign and 34 malignant) from the Department of Obstetrics and Gynecology, Clinica Universidad de Navarra, University of Navarra School of Medicine, Pamplona, Spain (Center II), were enrolled into the study. RESULTS ADNEX achieved high accuracy in discriminating between malignant and benign ovarian tumors in both centers (79.9% and 81.3% in Centers I and II, respectively). Multiclass accuracy was substantially lower than in binary classification (malignant vs. benign): 64.2% and 74.0% in Centers I and II, respectively. Sensitivity and specificity for the diagnosis of specific tumor types in Center I were as follows: benign tumors - 72.4% and 94.3%; borderline tumors - 33.3% and 87.0%, stage I ovarian cancers - 00.0% and 91.8%; stage II-IV ovarian cancers - 68.2% and 83.1%; and metastatic tumors - 00.0% and 99.5%. Sensitivity and specificity in Center II were as follows: benign tumors - 75.3% and 97.1%; borderline tumors - 50.0% and 88.2%, stage I ovarian cancers - 40.0% and 97.5%; stage II-IV ovarian cancers - 95.0% and 88.3%; and metastatic tumors - 20.0% and 98.3%. CONCLUSIONS ADNEX is characterized by very high accuracy in differentiating between malignant and benign adnexal tumors. However, prediction of ovarian tumor types could be more accurate.

Senescent peritoneal mesothelium creates a niche for ovarian cancer metastases

Although both incidence and aggressiveness of ovarian malignancy rise with age, the exact reason for this tendency, in particular the contribution of senescent cells, remains elusive. In this project we found that the patient’s age determines the frequency of intraperitoneal metastases of ovarian cancer. Moreover, we documented that senescent human peritoneal mesothelial cells (HPMCs) stimulate proliferation, migration and invasion of ovarian cancer cells in vitro, and that this effect is related to both the activity of soluble agents released to the environment by these cells and direct cell-cell contact. The panel of mediators of the pro-cancerous activity of senescent HPMCs appeared to be cancer cell line-specific. The growth of tumors in a mouse peritoneal cavity was intensified when the cancer cells were co-injected together with senescent HPMCs. This effect was reversible when the senescence of HPMCs was slowed down by the neutralization of p38 MAPK. The analysis of lesions excised from the peritoneum of patients with ovarian cancer showed the abundance of senescent HPMCs in close proximity to the cancerous tissue. Collectively, our findings indicate that senescent HPMCs which accumulate in the peritoneum in vivo may create a metastatic niche facilitating intraperitoneal expansion of ovarian malignancy.

Oxidative stress contributes to hepatocyte growth factor-dependent pro-senescence activity of ovarian cancer cells

Abstract The cancer‐promoting activity of senescent peritoneal mesothelial cells (HPMCs) has already been well evidenced both in vitro and in vivo. Here we sought to determine if ovarian cancer cells may activate senescence in HPMCs. The study showed that conditioned medium (CM) from ovarian cancer cells (OVCAR‐3, SKOV‐3, A2780) inhibited growth and promoted the development of senescence phenotype (increased SA‐&bgr;‐Gal, &ggr;‐H2A.X, 53BP1, and decreased Cx43) in HPMCs. An analysis of tumors isolated from the peritoneum of patients with ovarian cancer revealed an abundance of senescent HPMCs in proximity to cancerous tissue. The presence of senescent HPMCs was incidental when fragments of peritoneum free from cancer were evaluated. An analysis of the cells’ secretome followed by intervention studies with exogenous proteins and neutralizing antibodies revealed hepatocyte growth factor (HGF) as the mediator of the pro‐senescence impact of the cancer cells. The activity of cancerous CM and HGF was associated with an induction of mitochondrial oxidative stress. Signaling pathways involved in the senescence of HPMCs elicited by the cancer‐derived CM and HGF included p38 MAPK, AKT and NF‐&kgr;B. HPMCs that senesced prematurely in response to the cancer‐derived CM promoted adhesion of ovarian cancer cells, however this effect was effectively prevented by the cell protection against oxidative stress. Collectively, our findings indicate that ovarian cancer cells can elicit HGF‐dependent senescence in HPMCs, which may contribute to the formation of a metastatic niche for these cells within the peritoneal cavity. Graphical abstract Figure. No caption available. HighlightsConditioned medium (CM) from ovarian cancer cells induces senescence in HPMCs.Hepatocyte growth factor (HGF) mediates the pro‐senescence activity of cancerous CM.Activity of cancerous CM/HGF is associated with the induction of oxidative stress.Signaling pathways underlying activity of CM/HGF include AKT, p38 MAPK and NF‐&kgr;B.HPMCs senesced in response to cancerous CM/HGF promote ovarian cancer cell adhesion.

An Intelligent System for Computer-Aided Ovarian Tumor Diagnosis

This article describes the fundamentals of an intelligent decision support system for the diagnosis of ovarian tumors. The system is designed to support diagnosis by less experienced gynecologists, and to gather data for continuous improvement of the quality of diagnosis. The theoretical basis for the construction of the system is the IF-sets framework, used to aggregate multiple decision-making methods, and simultaneously providing information about positive and negative diagnosis of a given tumor type.

Extracellular matrix metalloproteinase inducer (EMMPRIN) expression correlates positively with active angiogenesis and negatively with basic fibroblast growth factor expression in epithelial ovarian cancer

PurposeThe primary aim of this paper was to evaluate the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its relationship with proangiogenic factors and microvessel density (MVD) in ovarian cancer.MethodsThe study group included 58 epithelial ovarian cancers (EOCs), 35 benign ovarian tumors, and 21 normal ovaries. The expression of EMMPRIN, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) was assessed by ELISA of tissue homogenates. Antibodies against CD105, CD31, and CD34 were used to immunohistochemically assess MVD.ResultsWe have found significantly higher EMMPRIN expression in EOC than in benign ovarian tumors and normal ovaries. Similarly, the VEGF expression was higher in EOC than in benign ovarian tumors and normal ovaries. By contrast, bFGF expression was lower in EOC than in benign ovarian tumors and ovary samples. EMMPRIN expression in EOC was directly correlated with VEGF expression and CD105-MVD, but inversely correlated with bFGF expression. Grade 2/3 ovarian cancers had increased expression of EMMPRIN and VEGF, increased CD105-MVD, and lowered expression of bFGF compared to grade 1 ovarian cancers. Moreover, EMMPRIN expression was higher in advanced (FIGO III and IV) ovarian cancer.ConclusionsThe upregulation of EMMPRIN and VEGF expression is correlated with increased CD105-MVD and silenced bFGF, which suggests early and/or reactivated angiogenesis in ovarian cancer. Aggressive EOC is characterized by the following: high expression of EMMPRIN and VEGF, high CD105-MVD, and low expression of bFGF.

Biochemical composition of malignant ascites determines high aggressiveness of undifferentiated ovarian tumors

Although undifferentiated tumors are the most lethal among all ovarian cancer histotypes, the exact reasons for this situation are unclear. This report was aimed at investigating whether the high aggressiveness of undifferentiated ovarian cancer may be associated with a biochemical composition of malignant ascites accumulating in the peritoneal cavity. We analyzed ascites from patients with undifferentiated, high-grade serous, endometrioid and clear-cell ovarian cancers, and from non-cancerous patients with respect to a group of soluble agents involved in cancer cell progression. Moreover, the effect of these fluids on proliferation and migration of ovarian cancer cells (A2780, OVCAR-3 and SKOV-3) was evaluated. The study showed that the level of all tested proteins in malignant ascites was higher than in the benign fluids. Concentration of 9/11 agents (CCL2, CXCL1, CXCL5, CXCL8, CXCL12, HGF, PAI-1, TGF-β1 and VEGF) was the greatest in the fluids from undifferentiated cancer, while the level of remaining 2 (IL-6 and uPA) was the highest in ascites from serous carcinoma. Proliferation of cancer cells was the most effective when they were subjected to ascites from patients with undifferentiated and serous cancer, whereas the migration was the highest in the case of undifferentiated tumors. Our findings indicate that the aggressiveness of undifferentiated ovarian tumors may be associated with the composition of malignant ascites, in particular the concentration of specific proinflammatory, cancer-promoting agents.

Serum arylesterase and paraoxonase activities in patients with ovarian tumors

OBJECTIVE High levels of toxic reactive oxygen species have been found in many types of cancer cells. Serum arylesterase (ARE) and paraoxonase (PON) are esterase enzymes that have strong antioxidant characteristics. The main purpose of our study was to evaluate the activity of ARE and PON in the sera of patients with ovarian cancer and benign ovarian tumors. MATERIALS AND METHODS This study included 30 patients with ovarian cancer, 42 patients with benign ovarian tumors, and 19 healthy age- and sex-matched individuals. ARE and PON activities were measured using spectrophotometry. RESULTS Serum ARE activity was significantly different among the three studied groups (p<0.0001). However, posthoc tests revealed that ARE activity was lower in the benign ovarian tumor group (median, 1.53 U/mL; range, 0.43-2.47 U/mL) than in the other groups. There were no differences in ARE activity between patients with ovarian cancer (1.89 U/mL; range, 1.01-2.56 U/mL) and healthy individuals (2.05 U/mL; range, 0.79-2.44 U/mL). We found no differences in PON activity or the PON:ARE activity ratio between the studied groups. Tumor size in the benign ovarian tumor group was positively correlated with ARE activity (R Spearman=0.46, p=0.003) and negatively correlated with PON activity (R Spearman=-0.50, p=0.001). The ARE and PON activities were not influenced by histological type, ovarian cancer grade, or disease advancement. CONCLUSION ARE activity is higher in patients with ovarian cancer than in patients with benign ovarian tumors; however, the serum activity of ARE is similar between patients with cancer and healthy individuals.

The associations between serum VEGF, bFGF and endoglin levels with microvessel density and expression of proangiogenic factors in malignant and benign ovarian tumors.

AIM OF THE STUDY To investigate whether serum levels of VEGF, bFGF and endoglin correlate with tumor VEGF and bFGF expression or microvessel density (MVD) in ovarian cancer. PATIENTS AND METHODS Forty five patients with epithelial ovarian cancers (EOCs) and 38 patients with benign ovarian tumors (BOTs) were included into the study. Serum levels of VEGF, bFGF and endoglin were assessed using ELISA. The expression of VEGF and bFGF in tumor samples were evaluated using ELISA of supernatants obtained from tumor homogenization. MVD was analyzed using immunohistochemistry with antibodies against CD31, CD34 and CD105. RESULTS Serum VEGF levels were significantly higher in EOCs than in BOTs (436.6pg/ml [19.67-2860] vs 295.5pg/ml [123-539], P=0.025). Serum endoglin levels were lowered in the group EOCs when compared to BOTs (33,720g/ml [12,220-73,940] vs 42,390pg/ml [19,380-56,910], P=0.015). There were no differences in bFGF levels between studied groups. EOCs have significantly higher CD105 MVD (25 vessels/mm2 [0-57] vs 6 vessels/mm2 [0-70], P<0.001) and tumor VEGF (405.9pg/mg protein [0-3000] vs 2.225 [0-634.7], P<0.001) expression than BOTs, while, bFGF expression was higher in BOTs than in EOCs (2076pg/mg protein [668.1-8718] vs 847.3pg/mg protein [188.9-8333], P=0.003). In patients with EOCs we have observed negative correlation between serum VEGF concentration and its tissue expression (r Spearman=-0.571, P=0.0261), and serum VEGF concentration correlated positively with CD34-MVD (r Spearman=0.545, P=0.0289). In a multiple regression analysis we have observed only the negative correlation between serum VEGF and CD105-MVD (r=-0.5288, P=0.0427). CONCLUSIONS Serum VEGF is a useful marker for prediction of ovarian cancer MVD and tumor VEGF expression.