| Background:Epithelial ovarian carcinoma is the most lethal gynaecological malignancy. Despite optimal cytoreductive surgery and paclitaxel/platinum-based chemotherapy,70% of patients will have persistent or recurrent disease, and the 5-year survival rate in advanced patients is only 30%-40%. Resistance of tumor cells to chemotherapeutic drugs is one of the main causes of treatment failure. Understanding the mechanism and further predicting clinical platinum resistance are important to treat patients individually and eventually improve prognosis.Our research team has focused on platinum resistance study in ovarian cancer since 2003. Platinum resistant protein annexin A3 was selected by comparative proteomics. In vivo animal study, when cisplatin was administrated to nude mice xenografted with annexin A3 overpression tumors, the inhibition of tumor growth was significantly reduced. In vitro study, after transfecting with annexin A3-expressed plasmid into drug sensitive ovarian cancer cells, both intracellular platinum concentration and platinum-DNA binding capacity lowered. Annexin A3 could be detected in culture medium supernatant of ovarian cancer cell lines and serum of ovarian cancer patients. Further studies demonstrated that annexin A3 was secreted outside the cell by exosomes and at the same time platinum effluxion completed by binding to exosomes.Based on above results, the present study focused on translation from basic study to clinical application. Annexin A3 expression was detected prospectively in tumor tissue and peripheral blood of ovarian cancer patients. The correlation between annexin A3 expression and clinical platinum resistance was analysed. Meanwhile, possible mechanisms which affect annexin A3 protein expression and platinum resistance were explored preliminary.Methods:1,Epithelial ovarian cancer patients were enrolled into the study prospectively and continuously from Sep 2009 to Apr 2010, who were given primary cytoreductive surgery and platinum-based chemotherapy in PUMCH. The clinic data was collected in detail. The tumor tissues and peripheral blood samples before, during and after chemotherapy were collected continuously. Patients were followed-up in PUMCH and the primary endpoint was PFS and response to platinum-based chemotherapy.2,The expression of annexin A3 in tumor tissues of ovarian cancer patients was detected by immunohistochemistry method. The correlation between annexin A3 expression and platinum resistance was analysed by Logistic regression. The effection of annexin A3 expression on disease-free survival was analysed by Cox regression method.3,The expression of annexin A3 protein in peripheral blood of ovarian cancer patients during treatment was detected by ELISA method. The expression of annexin A3 in peripheral blood and in tumor tissue was compared. The correlation of annexin A3 expression and clinical platinum resistance was analysed.4,Ovarian cancer tissues were purified from formalin-fixed paraffin-embedded operational samples by manual microdissection. The methylation status of annexin A3 promoter in ovarian cancer tissues and ovarian cell lines were evaluated by methylation specific PCR (MSP) method. The methylation level between annexin A3 high expression group and low expression group was compared, also between platinum resistant group and sensitive group. Then the effection of different mthylation level on annexin A3 protein expression and platinum resistance were evaluated.Results1,Fifty-three epithelial ovarian cancer patients were enrolled into the study from Sep 2009 to Apr 2010. The median follow-up time was 15 months (range 4.4-20.2months). During follow-up period,4 patients were lost and 17 patients had progressive disease including 5 patients died. Among 49 patients,38 (77.6%) were platinum sensitive and 11(22.4%) platinum resistant.2,Annexin A3 expression was measured in 51 patients'tumor tissues by IHC. Among 51 samples,13(25.5%) showed negative,12(23.5%) weak positive,13(25.5%) moderate positive, and 13(25.5%) strong positive. Negative and weak positive were classified as annexin A3 low expression group, moderated and strong positive as annexin A3 high expression group. Annexin A3 expression was not related to the pathological type, tumor grade, or disease stage (p=0.499,0.610, and 0.703 respectively). Except 4 lost patients, only 1(4%) patient was platinum resistant in annexin A3 low expression group, while 10(45.5%) in annexin A3 high expression group (p=0.001). Annexin A3 overexpression increased the risk of platinum resistance significantly (OR=27.6,95%CI 2.3-327.3). Furthermore, patients in annexin A3 high expression group had a poorer prognosis compared with low expression group (mean PFS=13.9±1.3months vs 18.2±0.8months, p=0.01). By COX risk regression analysis, annexin A3 overexpression in tumor tissue affected the PFS significantly (RR=3.219,95%CI 1.126-9.206).3,Annexin A3 protein could be detected in peripheral blood of ovarian cancer patients and healthy women. The concentration before chemotherapy in ovarian cancer patients(0.325 ng/ml,0.157-1.25ng/ml) was higher than that in healthy women(0.187 ng/ml,0.058-0.323ng/ml) (p<0.001). The correlation of annexin A3 expression between peripheral blood and tumor tissue was analyzed. The serum annexin A3 concentration was higher in annexin A3 high expression group than in low expression group (0.373ng/ml vs.0.298ng/ml), and the concentration of positive group was higher than negative expression group(0.367ng/ml vs.0.273ng/ml). While there were no statistical difference because of small sample size (p=0.376 and 0.125).During the period of treatment, the concentrations of annexin A3 during chemotherapy (0.178 ng/ml,0.014-0.327) and after chemotherapy (0.136 ng/ml, 0.016-0.40) were significantly lower than that before chemotherapy (P<0.001). But the concentration of annexin A3 in patients with recurrent disease (0.250ng/ml) was higher than that during chemotherapy and after chemotherapy (p=0.003 and 0.001 repectively). Platinum sensitive group had the same trend. However in platinum resistance group, the concentration of during chemotherapy was 0.148ng/ml(0.064-0.285ng/ml), but the concentration of after chemotherapy raised to 0.188ng/ml(0.028-0.337 ng/ml).The concentration of annexin A3 in peripheral blood was significantly correlative to serum CA 125 level by Person correlation analysis (Pearson correlation coefficient=0.312, p<0.001). All the samples were divided into two groups according to serum CA125 level (≤35U/ml and >35U/ml), annexin A3 concentration of abnormal CA125 group was significantly higher than normal CA125 group (0.171 ng/ml vs.0.270ng/ml, P<0.001).4,The promoter of annexin A3 gene had CpG island by analysis using MSPPrimer software. The promoter of annexin A3 gene was in a status of mixed methylation and unmethylation in both ovarian cancer cell lines and tumor tissues. The median methylation level of tumor tissues was 33.3%(5.1%-86.5%). The correlation between methylation level and tumor tissue annexin A3 expression was analysed. The methylation level was higher in annexin high expression group (37.5%,6.1%-73.7%) than in low expression group (27.2%,5.1%-86.5%), while there was no statistical difference (p=0.559). There was also no difference between platinum sensitive group and platinum resistant group (31.7%(6.1%-79.3%) vs 38%(5.1%-64.1%), P=0.86).Conclusions1,The expression of annexin A3 protein in ovarian cancer tissue was significantly correlated with platinum resistance and prognosis. The patients with annexin A3 overexpression had higher risk of platinum-resistance and shorter PFS. The present study revealed that annexin A3 expression in ovarian caner tissue may act as a biomarker to predict clinical platinum resistance and prognosis.2,The expression of annexin A3 protein could be detected in both ovarian cancer patients and healthy women. The concentration before chemotherapy in ovarian cancer patients is much higher than that in healthy women. The concentration of annexin A3 in peripheral blood was positively correlative with the expression in tumor tissue, while there is no statistical difference because of small sample size. The concentration of annexin A3 in peripheral blood decreased significantly after chemotherapy in platinum sensitive group, while increased in platinum resistance group. Annexin A3 concentration increased obviously after disease recurrence. The concentration of annexin A3 in peripheral blood is positively correlative to serum CA125 level. The concentration of annexin A3 in abnormal CA125 group is higher than in normal CA125 group. These results indicated the predictive value of annexin A3 for platinum resistance in ovarian cancer. It is important to develop more stable and sensitive method to detect annexin A3 in peripheral blood of ovarian cancer patients.3,The methylation status of annexin A3 gene promoter was evaluated for the first time. The promoter of annexin A3 gene had CpG island, and annexin A3 gene was in a status of mixed methylation and unmethylation in both ovarian cancer cell lines and tumor tissues. The methylation level was not correlated to annexin A3 expression in tumor tissue or clinical platinum resistance.
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