Ovarian Cancer Of Platinum Resistance Marker Protein Annexin A3 Prospective Studies The Role Of Clinical Chemotherapy Drug Resistance Prediction

Background and PurposeEpithelia Ovarian cancer is the leading cause of gynecological cancer mortality. The standard treatment is satisfactory cytoreductive surgery and platinum-based combination chemotherapy, with taxol and carboplatin being the first line choice. In spite of the standard therapy the prognosis of EOC patients is not favorable and the5-year survival rate for advanced patients(stage Ⅲ and Ⅳ) is only30%. Chemoresistance has emerged as the major obstacle of failure in the treatment of ovarian cancer patients. The complete response rate of chemotherapy is less than75%and15-30%EOC patients does not response to the fist line regime. Among the cases displaying CR more than90%patients will suffer from recurrence. It is vital to predictive chemoresistance and improve the outcome of therapy. The current criterion of chemoresistance is based on the clinical chemotherapy outcome of chemotherapy and can not be detected before the determination of chemotherapy regime. Therefore, it is urgent to explore an effective predictive marker of platinum-resistance to guide the application of platinum before the treatment or at the early stage of chemotherapy.In previous studies of our research team, the platinum-resistance related protein Annexin A3was selected by comparative proteomics and further validated both in vitro and vivo. Further studies demonstrated that Annexin A3protein not only correlated with platinum resistance but also could be secreted outside the cells by exosomes which enabled Annexin A3as a potential serological platinum resistant biomarker. Due to the low sensitivity and poor reproducibility of Annexin A3enzyme-linked immunosorbent assay (ELISA) kit-the only commercially available kit, the Annexin A3protein chemiluminescence immunoassay kit(CLIA)) was newly invented by our team demonstrating high sensitivity and reproducibility. The diagnostic value of serological Annexin A3in the prediction of platinum resistance was verified in a prospective small sample clinical trial by plotting ROC curve. The area under the ROC curve was0.744and the best cutoff value was1.645ng/mL. At this point, the sensitivity, specificity, positive predictive value and negative predictive value were66.70%,82.93%,58.82%and87.18%respectively.Based on the previous study serum of epithelia ovarian cancer patients prior to chemotherapy were collected continuously. The level of Annexin A3protein was detected by CLIA and IHC and the predictive value of Annexin A3protein level was further assessed. Combined prediction model was explored to improve diagnostic efficiency. The significance of Annexin A3protein expression in relapsed epithelial ovarian cancer was also discussed.Methods1、Pre-treatment serum samples and clinical data were continuously collected. Annexin A3protein level was detected with CLIA kit and the distribution was observed. Follow the treatment outcome through clinical records and telephone interview. Analyze the relationship between serological Annexin A3protein level and clinical pathological factors by Spearman correlation analysis. Compare the Annexin A3protein concentration between platinum-sensitive and platinum-resistant group and analyze the relationship between Annexin A3protein concentration and chemoresistance in multivariate model. By building ROC curve evaluate the predictive value, determine the cutoff value and observe the sensitivity, specificity, positive predictive value and negative predictive value. Compare the ROC curve of current study with preliminary experiment. Analyze the effect of serological Annexin A3level on PFS by COX regression and the PFS between groups with different serological Annexin A3protein level was compared by Log rank test.2、Detect the Annexin A3protein expression by IHC and divide the cases into two groups, low-expression group(negative and"+") and high expression group("++" and "+++"). Compare the Annexin A3protein level by IHC between platinum-sensitive and platinum-resistant group and analyze the relationship between Annexin A3protein concentration and chemoresistance in multivariate model. Assess the predictive value of Annexin A3protein level by IHC on the prediction of chemoresistance, determine the cutoff value and observe the sensitivity, specificity, positive predictive value and negative predictive value. Compare the AUC of ROC curve of Annexin A3protein level by IHC with that detected by CLIA kit. Analyze the influence of serological Annexin A3level on PFS by COX regression and observe the difference of PFS between groups with Annexin A3protein high-expression and low expression groups by Log rank test. Analyze the effect of serological Annexin A3level on PFS by COX regression and the PFS between groups with different serological Annexin A3protein level was compared by Log rank test.3、SKOV3and SKOV3/CDDP cells were cultured adherently in DMEM (HG), COC1and COC1/CDDP cells suspended in RPMI1640. The IC50of resistant cells was tested by MTT in SKOV3/CDDP cell and by CCK-8in C0C1/CDDP cell to calculate resistance index. Collected the cells in logarithmic growth phase, test protein concentration after cell lysis by BCA and detect Cofilin-1protein expression by Western Blot. Immunofluorescence (IF) was used to detected Annexin A3location and expression. The cells were cultured in serum free medium and Western Blot was used to verify the existence of Cofilin-1protein in culture medium.4、Collect the CA125value before and after the first course chemotherapy of patients receiving no adjuvant chemotherapy, calculate the CA125change rate and observe the distribution. Analyze the relationship between CA125change rate and platinum resistance by multivariate analysis. Observe the AUC of ROC curve of CA125change rate in the prediction of chemoresistance. Build the Logistic regression model of combined prediction with CA125change rate and plot ROC curve to compare the AUC of of combined prediction and single prediction.5、Collect the serum of recurrent epithelial ovarian cancer patients before treatment continuously and conduce follow up visit of chemotherapy outcome. Detect the serological Annexin A3protein level of specimen with CLIA kit. Divide the patients into three groups by platinum free interval(<12months and≥12months) and observe the serum Annexin A3expression among groups. Analyze the serum Annexin A3concentration between platinum resistant and sensitive groups. Observed the Annexin A3concentration between progression and progression-free patients at defferent time after the completion of chemotherapy. Divide patients into high level and low level group by50th percentile and compare the PFS of two groups.Results1、Eighty-nine patients were referred to Beijing Peking Union Medical College Hospital from September2009to April2012. Detect the serological Annexin A3protein level of specimen with CLIA kit, with the CV within and between boar lower than15%and20%respectively. The histogram and P-P figure of89cases with chemotherapy outcome demonstrated the non-normal distribution of serum Annexin A3concentration(Z=1.475, P=0.026). The concentration ranged between0.20ng/ml to6.18ng/mL, with a median concentration of1.20ng/mL and interquartile range1.62ng/mL. Until March2013,3cases (3.13%) were lost and the median follow-up time was14months,68cases platinum resistant (including refractory) and21cases platinum sensitive. The mean serum Annexin A3level of platinum sensitive patients was1.33±0.91ng/mL, significantly lower than platinum resistance patients(2.28±1.69ng/mL)(P=0.001).Multivariate Logistic analysis showed that serum Annexin A3level and satisfactory cytoreductive surgery had significant influence on chemoresistance(P=0.028;0.016). The AUC of ROC curve of serum Annexin A3level was0.733and the cut off value was2.05ng/mL. The sensitivity, specificity, positive predictive value and negative predictive value were61.11%,80.88%,45.8%and88.7%respectively. The AUC of ROC curve was larger than the preliminary experiment. The median PFS of low level group was longer than high level group(14months vs.11.5months). Multivariate COX analysis showed that serum Annexin A3level (P=0.002) and satisfactory cytoreductive surgery (P=0.001) had significant effect on PFS. The deference of PFS between high level group and low level group was concluded to be obvious(P<0.05) by Log-rank test.2、Among the same89EOC cases,38(42.7%) showed negative,30(33.7%) weak positive,15(16.9%) moderate positive, and6(6.7%) strong positive.68cases (75.3%) showed low expression and21(24.7%) showed high expression. The Spearman analysis demonstrated that Annexin A3level by IHC correlated with platinum resistance(P=0.266, or=0.012).42.9%of platinum resistance patients displayed high expression, higher than17.6%of platinum sensitive cases(P=0.017).Multivariate Logistic analysis showed that Annexin A3expression by IHC(P=0.004) and satisfactory cytoreductive surgery(P=0.002) had significant influence on chemoresistance. The AUC of ROC curve of Annexin A3expression by IHC was0.664and the cut off value was "high expression" and the sensitivity, specificity, positive predictive value and negative predictive value were42.86%,82.35%,58.82%and87.18%respectively. The AUC of serum Annexin A3concentration was larger than that of Annexin A3concentration by IHC, but the significance was not obvious(0.773&0.664, P=0.544). The mean PFS of low level goup was longer than high level group (15.5±6.7vs12.4±4.6月).Multivariate COX analysis showed that serum Annexin A3level (P=0.001) and satisfactory cytoreductive surgery (P=0.004) had significant influence on PFS. There was obvious deference between PFS of high expression group and low expression group(P<0.05).3、SKOV3、SKOV3/CDDP、COC1and COC1/COD were stably cultured. The RI of SKOV3/CDDP to SKOV3and COC1/CDDP to COC1was6.40±1.55and2.87±1.16respectively. Cofilin1protein expression detected by Western blot in cytoplasm of SKOV3/CDDP and COC1/CDDP was higher than platinum sensitive cells and the deference was more obvious in SKOV3/CDDP and SKOV3cells. Annexin A3was observed to located in cytoplasm by Immunofluorescence (IF) and the platinum resistant cell had stronger fluorescence than sensitive cells. There was no Annexin A3expression detected in the concentrated serum free culture medium.4、61cases who received no neoadjuvant chemotherapy were included into analysis,48platinum sensitive and13platinum resistant. The CA125decrease rate displayed nonnormal distribution(Z=2.892; P<0.001). The CA125decrease rate of the platinum resistant was significantly lower than that of sensitive cases(P=0.001). After exclusion of one extreme value multivariate analysis of60cases concluded that serum Annexin A3level(P=0.017) and CA125decrease rate(P=0.012) influence the chemoresistance obviously. The AUC of CA125change rate was0.813and the cut off value was57.7%. The sensitivity, specificity, positive predictive value and negative predictive value were92.31%,61.70%,40.0%and96.7%. build the Logistic regression model of chemoresistance risk with serum Annexin A3level and CA125decrease rate and plot the ROC curve of combined prediction. The AUC of combined prediction was0.863and the sensitivity, specificity, positive predictive value and negative predictive value were92.31%、74.47%、50.0%and97.2%. The AUC of combined prediction was obviously larger than that of serological Annexin A3concentration.5-, Sixty-one relapsed EOC patients were included,54cases PFI>6months and7cases PFI<6months.17cases platinum resistant(including nine refractory patients) and7cases sensitive. Detect the serological Annexin A3protein level of specimen with CLIA kit, the CV within and between boar lower than15%and20%respectively. The histogram and P-P figure of serological Annexin A3protein level with chemotherapy outcome demonstrated normal distribution (Z=1.265, P=0.082) and the mean concentration was0.77±0.56ng/mL. The Annexin A3protein level of short PFI patients(≤12months) was higher than long PFI patients(>12months)(0.92±0.60ng/ml vs0.67±0.50ng/ml,P=0.08). The A3protein concentration of platinum resistant patients was high than platinum sensitive cases although the significant was not obviousd(0.87ng/mL vs0.67ng/mL, P=0.314). Compared with progression-free patients progression cases had higher Annexin A3protein concentration at defferenttime after completion of chemotherapy although the deference was not obvious.There was no significant difference between PFS of Annexin A3protein high level and low level patients (P=0.09)Conclusions1、Serological level of EOC patients before treatment was significantly correlated with platinum resistance and prognosis. As a serological predictive marker Annexin A3has medium value in the prediction of platinum resistance and the cut-off value is2.05ng/mL。2、The Annexin A3protein measured by IHC in EOC patients has significant correlation with platinum resistance and prognosis and the value as a platinum resistance predictive marker is concluded to be low. The AUC of serological Annexin A3protein concentration is larger than Annexin A3protein measured by IHC although there is no significant deference. 3、In vitro experiments confirmed that cofilin-1protein correlates with platinum-resistance, but has no extracellular secretion and therefore can not act as a platinum-resistant serological predictive marker.4、The CA125decrease rate has significant correlation with platinum resistance; Combined detection with serological Annexin A3protein concentration and CA125decrease rate has superior value than single factor in the prediction of platinum.5、The small sample preliminary study concluded that serological Annexin A3protein level has no obvious relationship with chemotherapy response in relapsed ovarian cancer patients.