Tumor budding index and microvessel density assessment in patients with endometrial cancer: A pilot study.

The present study aimed to analyze the association between tumor budding index (TBI) and microvessel density (MVD) and selected clinicopathological features in female patients with endometrial cancer (EC). The present study included 137 patients, of whom 117 had endometrial endometrioid cancer and 3 had non-endometrioid EC (NEEC). Additionally, 8 cases of simple endometrial hyperplasia and 9 cases of atypical endometrial hyperplasia were included in the present study. Patient age, menopausal status, tumor histological type, grade and International Federation of Gynecologists and Obstetricians (FIGO) clinical stage were investigated. Immunohistochemistry was utilized to detect MVD using a CD34 antibody, and a laminin-5γ2 antibody was used for TBI assessment. In nonmalignant endometrial lesions, the TBI was significantly lower than that in patients with EC and NEEC (P=0.002). Significant differences in median TBI (MD-TBI) were also observed between patients with low-grade EC (MD-TBI, 4.5) and high-grade EC (MD-TBI, 16.2; P=0.01). Age, body mass index and tumor FIGO stage were not indicated to be associated with the MD-TBI. Premenopausal patients with EC had lower MD-TBI values than postmenopausal patients (0.3 vs. 11.1; P<0.005). The median MVD-CD34 in the study group was 19 (range, 13-29). Significant differences in MVD-CD34 were observed between malignant and nonmalignant endometrial lesions (P=0.01). Histological grade was markedly associated with tumor MVD-CD34 (P=0.001). The MVD was higher in high-grade cancer (G3; MVD-CD34, 24.9) than in grade G1 and G2 lesions (MVD-CD34, 14 and 18.6, respectively; P=0.01). FIGO clinical stage was not associated with MVD-CD34 in low and high stage lesions (MD, 18.4 for FIGO stage I/II; MD, 17.6 for FIGO stage III/IV; P=0.2). High MVD was markedly associated with high MD-TBI (P=0.0002). In conclusion, TBI could be a valuable indicator of tumor aggressiveness in patients with EC. The presence of the tumor budding phenomenon with increased MVD may have the potential to further refine clinical management decisions when endometrial malignancy is detected.

Prognostic significance of TEM7 and nestin expression in women with advanced high grade serous ovarian cancer.

OBJECTIVES: Tumor endothelial marker 7 (TEM7) and nestin have been proposed to be new candidates for neoangiogenesis assessment. Nestin is also cancer stem cells marker in various malignant tumors. AIMS: To investigate the expression of TEM7, nestin and nestin-related microvessel density (MVD) in high-grade serous ovarian cancer samples and to study their correlation with overall survival (OS) and disease-free survival (DFS) times. MATERIAL AND METHODS: Tumor samples obtained from 70 women with FIGO IIIc/IV ovarian serous cancer were studied with immunohistochemistry. RESULTS: Patients median age was 54 yrs (range: 29-72 years), 86% died of the disease with median OS = 28.5 months and median DFS = 10 months (3 years DFS = 19%; 5 years. DFS = 13.8%). High nestin expression was found in 16 (23%) patients with 3 years and 5 years OS of 14% and 0%. In low-nestin expression group OS and DFS were 42% and 25%, respectively. Median nestin-MVD (16, range:12-23) was not correlated with cancer cells nestin expression and with both DFS and OS. High TEM7 expression was found in 29 women (41%) of whom 21 (72%) died of the disease. A 5-year OS in these women was 27% as compared to 8% in low TEM7 expression group, but TEM7 presence had no association with nestin, nestin-MVD and both OS and DFS. CONCLUSIONS: Nestin as a marker of cancer stem cells may assist in the prediction of OS and DFS in women with high grade serous ovarian cancer. Nestin may also be considered a novel therapeutic target for antiangiogenic agents.

Mig-7 expression and vasculogenic mimicry in malignant ovarian tumors.

OBJECTIVES: To investigate the possible association of vasculogenic mimicry (VM), VE-cadherin and MIG-7 expression with clinicopathological features of women with malignant ovarian masses. MATERIAL AND METHODS: VM was studied with the PAS reaction and VE-cadherin was assessed with immunohistochemistry in 108 women with malignant ovarian tumors. Additionally, quantitative expression of MIG-7 mRNA was performed in 52 ovarian cancers with qRT-PCR. RESULTS: VM was found in 48/108 cases (44%), more often in higher FIGO stage tumors (83% cases; 40 vs. 8; p = 0.01). High expression of VE-cadherin was present in 37% of all ovarian masses. Ovarian tumors without VM more often expressed low levels of VE-cadherin than tumors where VM was found (37.6% vs.14.6%). No expression or very low expression of MIG-7 mRNA was found in all normal ovarian tissues and in 32 cancer samples. Median RQ of MIG-7 mRNA in tumor samples was higher than in normal ovarian tissue (RQ = 0.29 vs. RQ = 0.05, respectively; p < 0.005) and higher than in non-malignant ovarian masses (0.98 vs. 0.05 respectively; p = 0.03). Expression of MIG-7 mRNA was significantly correlated with VM (p = 0.039). In tumors with PAS-positive structures median RQ MIG-7 mRNA was higher than in tumors with PAS-negative findings (1.89 vs. 0.13 respectively). VE-cadherin expression was more frequently found in tumors where MIG-7 mRNA was present (p = 0.004). CONCLUSIONS: Vasculogenic mimicry exists in malignant ovarian tumors and advanced clinical stages of malignancy are accompanied by a high incidence of VM formation. MIG-7 mRNA and VE-cadherin expression may serve as additional molecular markers of VM in ovarian malignancies.

Proliferation and maturation of intratumoral blood vessels in women with malignant ovarian tumors assessed with cancer stem cells marker nestin and platelet derived growth factor PDGF-B.

OBJECTIVES: Platelet-derived growth factor B (PDGF-B) and nestin have been suggested to be useful in the assessment of neoangiogenesis in malignant ovarian masses. We aimed to investigate a possible association of these markers with newly formed microcapillaries and perivascular cells in ovarian tumors. MATERIAL AND METHODS: Microvessel density (MVD) and pericytes were studied in 82 women with ovarian neoplasms, including 7 benign cysts, 7 borderline masses, 64 epithelial ovarian cancers and 4 other malignant ovarian tumors. Immunohistochemical staining included antibodies to CD34, PDGF-B and nestin. RESULTS: Median values of CD34-positive and nestin-positive MVD were: 24,5 (range:17-32) and 21 (range: 12-31), respectively. No significant correlation between intratumoral CD-34 positive MVD and nestin-positive MVD was found. Benign and borderline lesions more frequently than malignant tumors displayed low or medium values of nestin-positive MVD (p = 0.01). Histological grading of malignant tumors was associated with nestin-positive MVD (p = 0.01). Nestin expression in tumor cells was not correlated with tumor grade or histological subtype. PDGF-B expression was found in tumor microves-sels in 72% of cases (59/82). High expression of PDGF in pericapillary cells was strongly associated with high expression of this marker in cancer cells (p = 0.007). Significant correlations between PDGF-B and nestin expression in malignant tumor microvessels were also found (p = 0.04). Nestin and PDGF-B expressions were strongly associated with high grade tumors when compared to low grade or benign masses. CONCLUSIONS: We conclude that the assessment of PDGF-B and nestin-positive MVD could be used to identify only highly active, angiogenic malignant ovarian masses, where tumor vasculature is formed.

Opioid-binding protein/cell adhesion molecule-like (OPCML) gene and promoter methylation status in women with ovarian cancer.

OBJECTIVE: The expression profile of the OPMCL gene was studied to find out if there was any evidence of a CpG island methylator phenotype and if there was an association of CpG island methylation with the gene downregulation in women with ovarian cancer. MATERIAL AND METHODS: Expression of OPCML in 43 ovarian cancer tumor samples and in 4 normal ovaries was determined by RT-PCR. Methylation status of OPCML promoter region was studied with methylation-specific PCR (MSP) method. Possible associations with selected clinicopathologic variables: FIGO stage, histological grade, patient's age and menopausal status were tested. RESULTS: In all normal ovarian samples OPCML mRNA was present, but it was not detectable in 24 of 43 ovarian cancer cases. We have not found the relationship between age and menopausal status with the presence of RTPCR product of OPCML. Hypermethylation of OPCML was not correlated to FIGO stage, however, in 80% of cases with methylated OPCML early clinical stage was also present. Tumor grading and histological type had no significant influence on the presence of hypermethylation of OPCML gene. In 20 of 43 cases of ovarian cancer methylated product of MSP amplification was present. In a group of OPCMLmRNA-negative tumors there were 75% of cases with hypermethylated exon of OPCML and the correlation between these variables was statistically significant (chi2 =17.7; p=0,00003). No promoter hypermethylation of the studied gene was found in normal ovaries. CONCLUSIONS: Reduced OPCMPL gene expression in ovarian cancer in comparison to normal ovaries could be related to the hypermethylation of promoter region. This epigenetic alteration may be the reason of gene silencing and the loss of suppressor function.

The role of CpG islands hypomethylation and abnormal expression of neuronal protein synuclein-gamma (SNCG) in ovarian cancer.

BACKGROUND: The synucleins are a family of small cytoplasmic proteins that are expressed predominantly in neurons. Recently, their expression has been found also in many human cancers. AIM: To understand the molecular mechanisms underlying the abnormal expression of SNCG in malignant ovarian tumors, in this study, we examined the methylation status of a CpG island located in exon 1 of SNCG gene in a panel of ovarian malignant tumors to determine if DNA methylation is related to clinical and histological tumor characteristics. METHODS: SNCG mRNA expression in tumor samples was assessed by RT-PCR. Methylation status of SNCG gene was studied using methylation specific PCR (MSP). RESULTS: Study group included 43 ovarian carcinoma samples (40 primary and 3 metastatic). The expression of SNCG mRNA was detectable in 33 of 43 ovarian cancer cases (76.7%). There were no significant differences in the mRNA expression between serous or nonserous tumors. The presence of SNCG mRNA in tumor samples was not correlated with age and menopausal status of patients, also no correlation was found with clinical stage or histological grading of malignant tumors. In 29 of 43 (67.4%) cases of tumors unmethylated product of MSP amplification was present. In a group of SNCG mRNA-positive tumors there were 75.7% (25 of 33) cases with demethylated or hypomethylated exon 1 of SNCG. The differences between the groups were statistically significant (chi2 =4.46; p=0.034). Demethylation of SNCG was not related to clinical tumor stage (p>0.05), but it was strongly associated with tumor grading (chi2=6.66; p=0.035). Aberrant methylation of SNCG was more often seen in tumors of women after menopause (78.2% vs 55%). In postmenopausal women 18 of 33 (62.1%) tumor samples synuclein-gamma mRNA expression was found, however the differences were not statistically significant. No correlations between SNCG hypomethylation and patient age, clinical stage and tumor grading were found. In 9 of 43 samples (21%) both products of amplification with methylated or unmethylated primer sets were found. In all of these cases SNCG mRNA was present. CONCLUSIONS: SNCG mRNA is expressed in a substantial proportion of malignant ovarian tumors and demethylation is an important event in abnormal synuclein-gamma expression in most of these cases.