Study On Clinical Observation And Mechanism Of Bevacizumab In The Treatment Of Advanced Epithelial Ovarian Cancer

BACKGROUND Ovarian cancer, as the third requency of gynecologic malignancies, holds on the third incidence and the highest mortality in the female reproductive system.Moreover, the present situation is increasingly worse and worse. Generally, majority of the patients with early ovarian cancer does not show obvious symptoms,because of it’s deep anatomical location in pelvis, and ovarian cancer is lack of specific tumor markers, so the advanced,ovarian cancer is usually diagnosed. At present, the standard therapeutic project of ovarian cancer is surgery combined with platinum-based chemotherapy. Satisfactory surgical procedures are capable of removing the most macroscopic tumor, but it’s hard to overcome some metastases. In addition, these patients are prone to platinum-resistant. Together these factors determine the difficult treatment, more recrudescence and high mortality. Ovarian cancer 5-year survival is only about 30%.Therefore, it’s necessary to find a promising approach for the treatment of ovarian cancer. More and more researches have been focusing on the molecular target therapy in the different tumors, ovarian cancer is no exception.As a vascular endothelial growth factor(VEGF) inhibitor, Avastin(bevacizumab, Bev) is a recombinant humanized monoclonal anti –VEGF, and takes effect to inhibit endothelial cell proliferation and angiogenesis via binding endogenous VEGF receptors competitively to block downstream intracellular signal transduction, then result in the degradation of tumor vessels and reduction of vascular permeability. Therefore, the significance of molecular targeted therapy with Bevacizumab is attracting more attention in ovarian cancer. To investigate the therapeutic effects and safety of bevacizumab in clinic, our institute(Department of obstetrics and gynecology, Xi Jing hospital, Fourth Military Medical University) have treated 45 patients of ovarian cancer with chemotherapy combined with bevacizumab, followed by detection of VEGF expression and micro vessel density(MVD)change. In addition, to probe the pathological significance of VEGF and CD34 in ovarian cancer, and calculate the MVD,tissues of 98 cases(Normal ovary: 20, pure chemotherapy: 50 and I, II operation group: 28) were employed to analyze the expression and the relevance of MVD and VEGF.In 2004, FDA has approved BEV as a molecular targeted therapy for the metastatic colorectal cancer. Bevacizumab has been widely used as an effective therapy in lung, breast, colon, and other tissue cancers. Since 10 years ago, while ovarian cancer treated with Bevacizumab is undergoing Ⅱand Ⅲ phase of clinical trials abroad. Based on the positive results of GOG0218 and ICON7, European Medicines Agency EMA(European Medicines Agency, EMA) has authorized that Bevacizumab can be used the first-line therapy for the treatment of advanced ovarian cancer. The approval of more than one option for restricted to operation and chemotherapy for patients with ovarian cancer. However, little clinical research regard Bevacizumab applied in ovarian cancer is found in our country.Recent study has demonstrated that BEV treatment can not affect cell viability of ovarian cancer cell line SKOV3 from CCK-8 assay. But BEV can effectively reduce the proliferation and increase the apoptosis of SKOV3 in vitro. However, there is little research on influence of BEV in ovarian cancer cell.In this experiment, groups including imaging data with ovarian cancer patients, change values of serological CA125, paraffin sections of ovarian cancer and cell line SKOV3,were used statistical analysis the clinical use of bevacizumab in ovarian cancer patients after treatment, followed by detecting the expression of each group of ovarian cancer tissue VEGF protein with immunohistochemistry, in addition to observing the expression of CD34 protein and calculating micro vessel density(MVD). Together,to analyze the correlation between VEGF and MVD and analyze the relationship between their expression and clinic pathological possibilities. After selected the appropriate concentration of bevacizumab and cisplatin in SKOV3 cells, CCK-8(Cell Count Kit-8) was carried out to detect ovarian cancer SKOV3 cell proliferation after 24 h and 48 h respectively, flow cytometry was employed to test the apoptosis of SKOV3 cells treated with bevacizumab after 24 h,then the appropriate drug concentration(bevacizumab 150μg/ml, cisplatin 3μg/ml) was selected. Combination with bevacizumab and cisplatin CCK-8 was used to analyze the proliferation of SKOV3 cell at 24 h, and 48 h, while flow cytometry was used to detect the apoptosis of SKOV3 cell at 24 h. At last, we investigated the biological effects of bevacizumab on ovarian cancer cells and tissues.OBJECTIVES 1.To observe the therapeutic effects of bevacizumab in the patients with Epithelial ovarian cancer. 2.Investigation of the VEGF expression in ovary carcinoma, and micro vessel density(MVD) using CD34 labeled endothelial cells. Together,to analyze the correlation between VEGF and MVD and analyze the relationship between their expression and clinic pathological possibilities. 3. To detect the influence of combination bevacizumab and cisplatin on theproliferation and apoptosis in ovarian cancer cell line SKOV3. 4.Research on the inhibitory effects of bevacizumab in combination with cisplatin in human ovarian cancer cell line SKOV3.METHODS AND CONTENTS 1.Recent clinical results observed with chemotherapy in epithelial ovarian cancer after combined bevacizumab treatment, and evaluation of bevacizumab clinical safety. 2.VEGF and MVD in the group were in the normal ovary, early ovarian cancer group, chemotherapy, monoclonal antibody and chemotherapy in ovarian tissue expression in paraffin sections + Bev group were compared. 3.Different concentrations of Bev(50μg/ml, 100μg/ml, 150μg/ml, 200μg/ml), and the use of different concentrations of cisplatin(1.5μg/ml, 3μg/ml, 6μg/ml, 9μg/ml) alone in SKOV3 cells, dosing 24 h, 48 h later by CCK-8 assay proliferation of SKOV3 cells; by flow cytometry Bev group SKOV3 cell apoptosis rate 24 h. 4.Select the appropriate drug concentration(Bev 150μg/ml, cisplatin 3μg/ml), both joint administration, dosing 24 h, after 48 h proliferation assay with CCK-8 cells; by flow cell apoptosis rate detected 24 h. 5.Concept Chabei Bev in combination with cisplatin in ovarian cancer cell line SKOV3.RESULTS: 1.Recent clinical treatment effect observed changes in tumor markers-CA125 value, image changes and adverse reactions: All patients completed at least PT 6-8 cycles of chemotherapy, Bev combined with chemotherapy for at least 3-6 PT period, all patients evaluable term effect, serology CA125 values and adverse reactions. A total of 670 patients completed the cycle of chemotherapy, Bev is 183 times the frequency of use, with a median number of six. Currently survival was 8-16 months. PT term efficacy of chemotherapy in 50 patients after chemotherapy is: CR: 3 cases(6%), PR :27 cases(54%), SD: 12 cases(24%), PD: 8 patients(16%), objective response rate ORR:( 60%), disease control rate DCR:(84%). PT 45 cases of short-term effect after chemotherapy.Tony Bev for patients: CR: 7 cases(15.6%), PR: 28 cases(62.2%), SD: 7 cases(15.6), PD:3 cases(6.6%), objective response rate ORR(77.8%), disease control rate DCR(93.3%); appear Bev adverse reactions associated with rash and proteinuria, rash one case: 1/45(1.2%), rash disappeared after stopping. Proteinuria 2 cases:2/45(4.4%); serious adverse cerebral hemorrhage, thromboembolism, heart failure, gastrointestinal perforation does not appear, but the emergence of a common chemotherapy side effects, such as bone marrow suppression, nausea, vomiting, fatigue, hair loss and liver toxicity, the symptomatic relief of symptoms after treatment, patients can safely complete treatment. Two groups of patients in the chemotherapy group PT and PT + Bev treatment group, 10 times the mean continuous monitoring of tumor markers CA125 were compared, PT + CA125 mean epithelial ovarian cancer Bev treatment group compared with PT regimen decreased levels of CA125 values decline significantly.2.VEGF and MVD in normal ovarian group, early epithelial ovarian cancer group, chemotherapy group, and PT + Bev treated ovarian tissue expression in paraffin sections, respectively, compared to the situation: VEGF in normal ovarian group, I, II ovarian cancer group PT + chemotherapy group and positive expression of Bev treatment groups were as follows: 45%, 92.9%, 69.6% and 41.9%。Ovarian tissue VEGF expression was significantly higher than that in normal ovarian tissue(c2 = 13.5, p <0.05)。 ovarian cancer I, II ovarian cancer group, chemotherapy group and chemotherapy plus Bev in MVD value(51.2 ± 2.08, 34.5 ± 0.85, 16.67 ± 0.93)。Correlation of VEGF and MVD expression in epithelial ovarian carcinoma: the expression of VEGF expression and MVD were positively correlated。VEGF expression and MVD increased with tumor histological grade and lower。3.CCK-8 test results: Bev plus cisplatin group SKOV3 cell proliferation inhibition(P <0.05), the combined group of SKOV3 cell inhibition rate is greater than the individual treatment groups, Bev combined with cisplatin group of SKOV3 cell proliferation inhibition(P <0.05).Three groups of drugs on SKOV3 cell proliferation inhibition rate increased with increasing drug concentration, cisplatin, and the combined group increased with prolonged duration of action of the drug. Bev 24 hours after SKOV3 cell proliferation rate is higher than 48 hours later. To a certain extent, the three groups of drugs on SKOV3 cell proliferation inhibition rate showed concentration and time dependent. Interaction coefficient cisplatin and Bev two drugs: CDI <1, that is a synergistic effect between Bev and cisplatin, test results and flow cytometry showed: Bev and cisplatin combined with the use of pro-apoptotic effects of early SKOV3 cells than single drug effect. From this experiment, we can see: Bev in combination with cisplatin can reduce the dose of cisplatin, while reducing the toxicity of cisplatin, can prolong duration of action of drugs, Bev to cisplatin may have certain sensitizing effect.CONCLUSIONS 1.Bevacizumab in combination with chemotherapy can improve the clinical effects of advanced epithelial ovarian cancer in short-term with no significant adverse reaction and serious complications, in addition to thrombosis and gastrointestinal perforation. Furthermore, this approach has a good effect on ovarian cancer patientsdecline of serological CA125 value.2.The expression level of VEGF in ovarian cancer tissue was significantly higher than that of VEGF in normal ovarian tissue, VEGF expression decreased after bevacizumab in combination with chemotherapy.between bevacizumab in combination with PT chemotherapy group and chemotherapy group in addition, the expression of VEGF and MVD values decreased with the advanced tumor. A positive correlation between the expression of VEGF and MVD was found in ovarian carcinoma.3.Bevacizumab, Cisplatin and combination with the two approaches all can inhibit the proliferation of SKOV3 cells, but the agonistic role was significantly improved when bevacizumab in combination with Cisplatin was used.