卵巢癌铂类耐药标记蛋白Annexin A3化学发光试剂盒的研制及其在临床应用中的研究
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摘要
研究背景及目的
卵巢癌是死亡率居首位的女性生殖系统恶性肿瘤,肿瘤细胞对化疗药物产生耐药性是临床治疗的主要障碍。运用铂类耐药标记蛋白,早期发现肿瘤耐药,有助于临床及时调整化疗方案,改善患者预后。
本课题组前期运用比较蛋白质组学技术,发现并证实了一种卵巢癌铂类耐药标记蛋白膜联蛋白A3(Annexin A3),该蛋白能够以细胞内囊泡形成、转运、外泌体的形式外泌,在细胞培养液上清和人外周血中均可检测到不同水平的表达,AnnexinA3有望成为一种铂类耐药血清学标记物应用于临床。然而,在扩大样本量进行临床检测的研究中发现,本课题组所应用的目前市场上唯一可提供的ELISA试剂盒存在敏感度低、重复性差等突出问题,造成实验数据的准确性欠佳。因此,研制一种能高通量、稳定检测人外周血Annexin A3蛋白表达水平的免疫分析试剂盒成为本课题深入研究中亟待解决的关键问题。
化学发光免疫分析法(CLIA)是继放射免疫分析法、酶免疫分析法和荧光免疫分析法之后发展起来的一种新的免疫学测定方法,该技术将高特异性的抗原抗体反应与高灵敏度的化学发光反应结合起来,具有灵敏度高、特异性强、重复性好、操作简便、分析快速、无放射污染等优点,成为目前临床检测蛋白分子的主流方法。
本实验在前期有关卵巢癌铂类耐药特异性标记蛋白Annexin A3工作的基础上进行深入研究,旨在制备能够满足临床检测要求的Annexin A3蛋白化学发光试剂盒,并应用该试剂盒对不同女性人群外周血中的Annexin A3蛋白进行检测,以了解该蛋白在人外周血中的表达情况,初步确定其正常值范围,并探讨其作为血清学标志物对临床上卵巢癌患者铂类化疗耐药的预测价值。
研究方法
1、筛选匹配高特异性的Annexin A3抗原及抗体对,制备检测人外周血.中AnnexinA3蛋白的化学发光免疫分析试剂盒。
购买市售的人Annexin A3重组蛋白及六种Annexin A3多克隆、单克隆抗体作为检测抗原和抗体,化学发光免疫分析法检测不同组合的两种抗体与各浓度抗原反应的发光值,绘制标准曲线,计算线性拟合相关系数r及信噪比(最高浓度标准品与0值标准品发光值之比,S5/SO),r值越接近1、信噪比越大表明抗原抗体间的结合特异性越高,选择r值及信噪比最大的两种抗体作为制备试剂盒的捕获抗体和检测抗体。将人Annexin A3重组蛋白稀释成不同浓度作为标准品,将筛选出的捕获抗体包被在固相载体上,采用改良过碘酸钠法连接检测抗体和辣根过氧化物酶制备出酶标检测抗体,两种抗体与抗原形成双抗体夹心复合物,加入发光底物后,使该复合物发光,化学发光仪检测发光强度。交叉方阵滴定法确定捕获抗体与酶标检测抗体的最佳工作浓度,并分别采用一步法和两步法对反应模式进行优化,最终确立最佳检测体系,完成试剂盒的制备。
2、对制备完成的Annexin A3化学发光试剂盒进行灵敏度、精密度和稀释线性的评价,确定检测工作范围,并与本课题组前期研究中应用的目前市场上唯一可提供的Annexin A3酶联免疫吸附法(ELISA)检测试剂盒进行比较评价。
平行测量20个零值标准品(S0),以其发光值(RLU)的平均值(Mean)加上两倍标准差(SD)所对应的浓度值求得本方法的灵敏度。精密度指测定结果的可重复性,评价指标为变异系数(coefficient of variation, CV),分别用1.6ng/mL和0.8ng/mL两种浓度的Annexin A3蛋白作为高、低值质控品,每个质控品平行做20孔,用不同批次的包被板重复测3次,计算单次检测(批内)和多次检测(批间)的CV值,CV(%)=标准差(SD)／均数(Mean)。将1份本课题组前期实验中用ELISA法测定为高值的血清样品(浓度为1.25ng/mL),按1：1、1：2、1：4、1：8、1：16、1：32、1：64进行倍比稀释后测定发光值,对稀释倍数及发光值进行线性拟合,计算相关系数r。将各浓度的标准品重复检测10次,根据每次的标准曲线求得相应浓度值,计算10次测量的均数及标准差,进而求出各浓度点的变异系数CV,以CV为纵坐标,浓度值为横坐标,绘制精密度曲线图,选用图中变异系数小于10%的浓度范围作为试剂盒的工作范围。取健康女性、卵巢良性肿瘤、上皮性卵巢癌患者的血清样本各20份,分别应用两种试剂盒进行检测,比较测定值的分布及对低值血样的区分情况。
3、应用Annexin A3化学发光试剂盒高通量检测人外周血中Annexin A3蛋白的表达水平,确定其正常值范围,评估其作为卵巢癌铂类化疗耐药血清学标志物的可行性,同时与CA125比较在临床诊断卵巢癌耐药中的作用。
连续收集2009年9月至2012年4月期间在北京协和医院体检的健康女性血清、以及住院手术治疗的卵巢良性和交界性肿瘤患者术前血清、上皮性卵巢癌患者治疗前血清,应用CLIA试剂盒检测Annexin A3蛋白在各组人群外周血中的浓度,初步建立其正常参考值范围。前瞻性连续收集卵巢癌患者治疗前、术后化疗前、化疗过程中以及化疗结束后的血清样本,并记录同期的CA125测定值。应用CLIA试剂盒检测Annexin A3的浓度,评估各临床病理因素、手术及化疗对Annexin A3表达水平的影响。对所有卵巢癌患者进行随访,根据目前对卵巢癌铂类耐药的定义判断患者的化疗结局,分为敏感组和耐药组,明确Annexin A3在两组患者不同治疗时点的动态变化及表达差异。与CA125进行相关性分析,探讨各治疗时点的Annexin A3表达水平与铂类耐药的关系,通过诊断试验评估CA125及Annexin A3对铂类耐药的诊断或预测作用：制作受试者工作特征(ROC)曲线,计算曲线下面积(AUC)以及各浓度值对应的约登指数(又称正确诊断指数,等于敏感度与特异度之和减1),AUC=0.5表明无诊断价值,AUC在0.5～0.7时有较低准确性,在0.7～0.9时有一定准确性,在0.9以上有较高准确性,约登指数最大时表明以该对应浓度为界值进行诊断的准确性最高,因此选择该浓度值为最佳诊断界值,计算此时诊断的敏感度、特异度、准确度(总符合率)、假阳性率、假阴性率、阳性预测值及阴性预测值。通过COX风险回归模型及Kaplan-Meier生存分析评估治疗前Annexin A3水平对患者无进展生存期的影响。
研究结果
1、选用兔抗-Annexin A3多克隆抗作为捕获抗体,鼠抗-Annexin A3多克隆抗体作为检测抗体时,与各浓度Annexin A3抗原反应得到的标准曲线的相关系数r及信噪比均最大,分别为0.9982和19.56,故选择两者应用于试剂盒的制备。采用浓度为5μg/mL的捕获抗体包被固相载体,稀释度为1：250的酶标检测抗体进行检测时,与各浓度Annexin A3标准品反应得到的标准曲线的相关系数r及信噪比均最大,分别为0.9987和59.33,从而确定为最佳工作浓度。采用一步法和两步法检测时的相关系数均较好,分别为0.9983和0.9986,但一步法的信噪比略高于两步法(49.11vs45.57),结合操作的简便性,反应模式选择一步法。
2、试剂盒的最低检测浓度即灵敏度为0.08ng/mL;批内精密度为5.92～8.63%,批间精密度为5.15～6.42%；高值血样的稀释度与发光值间呈良好的线性相关,相关系数r为0.9997；标准曲线在0.1～1.6ng/mL浓度之间的变异系数均小于10%,因此选定0.1～1.6ng/mL作为工作范围。两种试剂盒检测结果显示,ELISA试剂盒对60份血样的中位测值为0.36ng/mL (0.1～2.03ng/mL),其中80%(48/60)的血样测值低于标准曲线建议的最低浓度(0.8ng/mL),30%(18/60)低于ELISA试剂盒说明书标明的最低检测浓度(0.2ng/mL); CLIA试剂盒对60份血样的中位测值为0.95ng/mL (0.04～2.77ng/mL),90%(54/60)的血样测值位于标准曲线范围内,其中2例低于试剂盒最低检测浓度(0.08ng/mL),4例高于标准曲线上限(1.6ng/mL),将该4份高值血清分别稀释2倍、4倍、8倍后进行复测,不同稀释倍数得到的最终测值间具有较好的一致性,变异系数均不到3%。CLIA试剂盒的操作步骤也较ELISA试剂盒简单,前者采用样本和酶标检测抗体同时加入的一步法检测模式,反应时间仅需2小时,而后者则需将样本和酶标检测抗体分三步加入,反应时间长达4.5小时。
3、共纳入符合标准的健康女性113例,卵巢良性肿瘤患者70例,卵巢交界性肿瘤患者14例,上皮性卵巢癌患者96例。四组人群外周血Annexin A3的中位浓度分别为0.75ng/mL (0.15～1.54ng/mL)、0.66ng/mL (0.04～1.79ng/mL)、0.65ng/mL (0.14～3.29ng/mL)以及1.12ng/mL (0.20～6.18ng/mL);卵巢癌患者治疗前Annexin A3水平明显高于健康女性及卵巢良性肿瘤患者(P值均为0.000),健康女性与卵巢良性肿瘤患者相比无统计学差异(P=0.322),卵巢交界性肿瘤患者与其余三组人群相比均无统计学差异(P值分别为0.144、0.132、0.236)。Annexin A3在113例健康女性外周血中的表达水平呈近似正态分布(Z=0.723,P=0.673>0.05),采用正态分布法确定参考值范围,正常值上限为Mean+1.96SD=1.45ng/mL
卵巢癌患者治疗前Annexin A3水平与年龄、组织学类型、组织学分级、手术病理分期、淋巴结转移均无关(P值分别为0.985、0.631、0.347、0.441、0.480)；术前及术后化疗前Annexin A3的中位浓度分别为1.11ng/mL (0.43～4.55ng/mL)、1.23ng/mL (0.4～4.32ng/mL),相比无统计学差异(P=0.226)；化疗前、化疗中、化疗后Annexin A3的中位浓度分别为1.15ng/mL (0.42～6.18ng/mL)、0.23ng/mL (0.02～3.00ng/mL)以及0.24ng/mL (0.03～1.82ng/mL),化疗中和化疗后较化疗前明显降低(P值均为0.000),化疗中与化疗后相比无统计学差异(P=0.487)。
截至2012年4月30日随访结束,96例卵巢癌患者的中位随访时间为10.73个月(2.23-32.17个月),共有56例可明确判断铂类耐药性结局,其中铂类敏感患者41例(占73.21%),耐药患者15例(占26.79%)；共有21例患者出现疾病进展,3例失访,3例仍在进行先期化疗,尚未手术。
敏感组患者治疗前、化疗中、化疗后的Annexin A3中位浓度分别0.80ng/mL (0.20～2.43ng/mL).0.31ng/mL(0.02～0.84ng/mL)、0.22ng/mL(0.06～0.75ng/mL),耐药组患者分别为2.21ng/mL (0.5～6.18ng/mL)、0.16ng/mL (0.04～0.76ng/mL)、0.24ng/mL (0.03～1.79ng/mL),两组患者化疗中及化疗后的Annexin A3水平均较化疗前明显降低(P值均为0.000),化疗中与化疗后相比无统计学差异(P值分别为0.605、0.296)；组间比较显示,耐药组患者治疗前Annexin A3水平明显高于敏感组(P=0.012),而化疗中与化疗后两组的Annexin A3水平无明显差别(P值分别为0.937、0.624)。
敏感及耐药患者治疗前CA125的平均浓度分别为(1096.51±1689.91)U/mL、(1896.48±2162.88)U/mL,两组比较无统计学差异(P=0.271),而耐药组患者化疗中CA125的表达水平明显高于敏感组(P=0.003),平均浓度分别为(16.52±12.47)U/mL、(198.89±527.32)U/mL,经相关性分析,治疗前、化疗中及化疗后AnnexinA3的表达水平均与CA125无关(P值分别为0.384、0.594、0.883)。治疗前CA125对铂类耐药无诊断作用,ROC曲线下面积AUC为0.597(P=0.271)；化疗中CA125诊断铂类耐药的AUC为0.755(P=0.014),最佳界值点为24.60U/mL,此时诊断铂类耐药的敏感度为60.00%,特异度为90.24%,准确度为82.14%,假阳性率为40.00%,假阴性率为9.76%,阳性预测值为69.23%,阴性预测值为86.01%。
以正常参考值上限1.45ng/mL为界对治疗前患者进行分组,高水平组发生铂类耐药的比率明显高于低水平组(55.56%vs13.16%,P=0.001),Logistic回归分析显示,治疗前Annexin A3水平是发生铂类耐药的危险因素之一(P=0.004,OR=9.241),高水平组发生铂类耐药的风险是低水平组的9.241倍。诊断试验结果显示,治疗前Annexin A3诊断铂类耐药的AUC为0.744(P=0.006),最佳界值点为1.645ng/mL此时治疗前Annexin A3预测铂类耐药的敏感度为66.70%,特异度为82.93%,准确度为78.57%,假阳性率为17.07%,假阴性率为33.30%,阳性预测值为58.82%,阴性预测值为87.18%。COX风险回归模型分析显示,治疗前Annexin A3水平是影响患者疾病进展的独立危险因素(P=0.001,RR=9.863),高水平组发生疾病进展的风险是低水平组的9.863倍,Kaplan-Meier生存分析显示,两组的中位PFS分别为27.60个月(2.23-32.17个月)和12.77个月(4.90-25.80个月),高水平组的PFS显著短于低水平组(P=0.001)。
结论
1、本研究从市售的六种Annexin A3多克隆、单克隆抗体中筛选出与人Annexin A3重组蛋白特异性结合最好的两种作为制备化学发光试剂盒的捕获抗体和检测抗体,首次成功制备出人Annexin A3蛋白化学发光定量检测试剂盒。
2、本研究制备的Annexin A3化学发光试剂盒具有较高的灵敏度,良好的重复性和稀释线性,操作方便、分析迅速,明显优于市售ELISA试剂盒,能够满足临床检测的要求,但该试剂盒还存在检测上限值偏低、高值血清样本需重新稀释后复测的缺点,需进一步改善以增加检测的线性范围。
3、应用本研究研制的化学发光试剂盒可以检测到不同人群外周血中Annexin A3蛋白的差异表达,卵巢癌患者治疗前Annexin A3水平明显高于健康女性及卵巢良性肿瘤患者,初步确定女性外周血Annexin A3的正常参考值上限。前瞻性观察56例可随诊结局的卵巢上皮癌患者,治疗前耐药组Annexin A3的的测定值明显高于敏感组,化疗期间耐药组CA125的的测定值明显高于敏感组,提示卵巢癌患者治疗前外周血Annexin A3的测定值对于临床出现铂类耐药具有一定的预测作用,化疗期间CA125测定值对于临床出现铂类耐药具有一定的诊断作用。此外,治疗前Annexin A3水平还是影响患者疾病进展的独立危险因素,是判断预后的重要指标。
Backgroud and Purpose
Ovarian cancer is the most lethal gynecological malignancy. Resistance of tumor cells to chemotherapeutic drugs is one of the main causes of treatment failure. Using a biomarker to predict clinical platinum resistance is important to treat patients individually and eventually improve prognosis.
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 also demonstrated that Annexin A3was a secreted protein. It could be secreted outside the cells by exosomes and be detected both in culture medium supernatant of ovarian cancer cell lines and serum of ovarian cancer patients. This characteristic enabled Annexin A3as a potential serological platinum resistant biomarker used in clinic. In order to explore the feasibility of quantitative detection of Annexin A3protein in the serum, our research team used the only commercially available detection kit—Annexin A3enzyme-linked immunosorbent assay (ELISA) kit. However, this kit was found the disadvantages such as low sensitivity, poor reproducibility and so on in subsequent clinical detection of large sample size. These outstanding problems resulted in poor accuracy of experimental data. Therefore, developing a new immunoassay kit for high-throughput and stably detecting the expression level of Annexin A3protein in human peripheral blood has become a key issue to be solved in our further study.
Chemiluminescence immunoassay (CLIA) is a new immunological detection method, which develops following radioimmunoassay, enzyme immunoassay and fluorescence immunoassay. It combines highly specific antigen-antibody reaction with high sensitivity chemiluminescent reaction, and has many advantages such as high sensitivity and specificity, good reproducibility, simple and convenient operation, without radioactive contamination and so on. CLIA has become the mainstream method in clinical detection of protein molecule.
Based on above background, the present study aimed to prepare the Annexin A3CLIA kit which can reach clinical detection requirements, and then applied it to quantitatively detect serum Annexin A3protein in different women population in order to understand its expression distribution in human peripheral blood, determine its normal range initially, and explore its predictive value for platinum chemoresistance in ovarian cancer patients as a serological marker.
Methods
1、Six kinds of commercially available anti-Annexin A3polyclonal or monoclonal antibodies were evaluated for their affinity with the antigen—human Annexin A3recombinant protein using double-antibody sandwich chemical luminescence enzyme immunoassay. The best pair was chosen as the capture antibody and detection antibody to prepare chemiluminescence immunoassay kit. Capture antibody was coated on microplate strips in different concentrations and detection antibody was conjugated with horseradish peroxidase (HRP) with modified sodium iodide method which was also diluted in different concentrations. They could form a double-antibody sandwich complex with Annexin A3antigen standard of different concentrations. Luminescence analyzer could detect the light intensity after adding light-emitting substrate which was proportional to the concentration of standard Annexin A3protein. The optimal working concentration of capture antibody and HRP labeled detection antibody was determined with cross-titration method for the best accuracy in measuring known concentrations of antigen. After optimizing the response mode, the final optimal detection system was established and the CLIA kit was prepared successfully.
2、Assess the Annexin A3CLIA kit in its sensitivity, precision (also called reproducibility) and dilution linearity, and determine the detection range by plotting the precision curve. The CLIA kit was also compared with the purchased ELISA kit by simultaneously detecting Annexin A3protein in sixty serum samples from healthy women and patients with benign ovarian tumor or epithelial ovarian cancer.
3、The serum specimens of healthy women at health checkup, benign or borderline ovarian tumor patients before surgery and epithelial ovarian cancer patients before treatment were collected continuously in Peking Union Medical College Hospital from September2009to April2012, and were detected by CLIA kits. The normal reference range of Annexin A3in healthy women was determined by95%confidence interval. The serum specimens of epithelial ovarian cancer patients before surgery or chemotherapy, after surgery but before chemotherapy, during chemotherapy and after chemotherapy were collected prospectively and continuously, all of which were detected by CLIA kits too. The relationship between the concentration of Annexin A3and the various clinicopathologic factors, surgery or chemotherapy was analyzed. All of the ovarian cancer patients were followed up in order to determine the chemotherapy outcome and disease status. According to the definition of clinical platinum chemoresistance, they were divided into the sensitive group and resistant group. The dynamic changes and expression differences of Annexin A3were analyzed in the two groups. By correlation analysis, the relationship between Annexin A3and CA125at different treatment points was evaluated. Diagnostic test was used to assess the diagnostic or predictive value of CA125and Annexin A3.Receiver operating characteristic (ROC) curve was drawn and the best diagnostic cutoff value was determined by the maximum Youden index which is equal to the sensitivity and specificity minus1, and then the diagnostic sensitivity, specificity, accuracy (total coincidence rate), false positive rate, the false negative rate, positive predictive value and negative predictive value were calculated. The ovarian cancer patients were also divided into the high-expression group and the low-expression group using the upper limit of normal for Annexin A3as cutoff value. The correlation between the Annexin A3level before treatment and progression free survival (PFS) was assessed by COX regression analysis and survival analysis.
Results
1、Using a rabbit anti-Annexin A3polyclonal antibody as capture antibody and a mouse anti-Annexin A3polyclonal antibody as detecting antibody to detect Annexin A3standard of different concentration showed the highest antigen-antibody binding specificity based on the largest correlation coefficient of the standard curve and the highest signal to noise ratio (SNR, the light intensity ratio of maximum value and zero value standard proteins),0.9982and19.56respectively. So this antibody pair was the best for preparing CLIA kit. The optimal working concentration of capture antibody and HRP labeled detection antibody was5μg/mL and1:250respectively which was also determined by the largest correlation coefficient0.9987and the highest SNR59.33. The reaction mode of one-step was better than two-step because of the similar correlation coefficient (0.9983vs0.9986) but higher SNR (49.11vs45.57) and easier to operate.
2、The sensitivity of Annexin A3CLIA kit was0.08ng/ml. The intra-assay precision was5.92～8.63%and the inter-assay precision was5.15～6.42%. It had good dilution linearity and the correlation coefficient of the fitting curve between sample dilution and light intensity was0.9997.The work scope of the kit was determined as0.1～1.6ng/ml for the coefficient of variation all less than10%. Detecting sixty blood samples showed the results as follows. The median measured value was0.36ng/ml (0.1～2.03ng/ml) by ELISA kit and0.95ng/ml (0.04～2.77ng/ml) by CLIA kit.80%(48/60) measured value were lower than the minimum recommended concentration (0.8ng/ml) of the standard curve, and30%(18/60) were lower than the minimum detectable concentration (0.2ng/mL) indicate by ELISA kit instructions. While using CLIA kit,90%(54/60) were in the range of the standard curve, only two lower than the minimum detectable concentration (0.08ng/ml). And the other four cases higher than the upper limit of the standard curve (1.6ng/ml) were re-tested by diluted two times, four times and eight times. It showed a good agreement among the final calculated values of different dilutions, and the coefficients of variation were all less than3%. The standard or samples were added at the same time in a one-step detection mode and the reaction time was only two hours. While the ELISA kit applied three-step mode by adding the standard or samples first, then detection antibody and last enzyme which made the reaction time up to4.5hours.
3、113healthy women,70patients with benign ovarian tumor,14patients with borderline ovarian tumor and96patients with epithelial ovarian cancer were enrolled into the study. The median concentration of serum Annexin A3in the four groups was0.75ng/ml (0.15～1.54ng/ml),0.66ng/ml (0.04～1.79ng/ml),0.65ng/ml (0.14～3.29ng/ml) and1.12ng/ml (0.20～6.18ng/ml) respectively. The concentration of Annexin A3before treatment in ovarian cancer patients was significantly higher than that in healthy women and patients with benign ovarian tumor (P=0.000). There was no significant difference between healthy women and patients with benign ovarian tumors (P=0.322). The level of Annexin A3had no significant difference in patients with borderline ovarian tumor compared with the other three groups respectively (P values were0.144,0.132and0.236). Kolmogorov-Smirnov test showed normal distribution of Annexin A3in healthy women (Z=0.723, P=0.673). The upper limit of normal for Annexin A3was defined as1.45ng/ml based on the mean value plus1.96standard deviations.
There was no correlation between the Annexin A3level before treatment and the age, histological type, histological grade, clinical stage, or lymph node metastasis in ovarian cancer patients (P values were0.985,0.631,0.347,0.441and0.480). The median concentrations of Annexin A3before and after surgery were1.11ng/ml (0.43～4.55ng/ml) and1.23ng/ml (0.4～4.32ng/ml) respectively, and there was no significant difference by comparison (P=0.226). The median concentration of Annexin A3before chemotherapy was1.15ng/ml (0.42～6.18ng/ml) and significantly reduced to0.23ng/ml (0.02～3.OOng/ml) during chemotherapy (P=0.000). After chemotherapy, the median concentration of Annexin A3was0.24ng/ml(0.03～1.82ng/ml), significantly lower than that before chemotherapy (P=0.000) but without significant difference compared with that during chemotherapy (P=0.487).
Up to April30,2012, the median follow-up time was10.73months (2.23to32.17months) in96patients with ovarian cancer, of whom56cases could be clearly judged the outcome of platinum chemoresistance, including41(73.21%) platinum-sensitive patients and15(26.79%) drug-resistant patients. During the follow-up period,3patients were lost,21patients had progressive disease, and3patients were still undergoing the neoadjuvant chemotherapy. The level of Annexin A3during chemotherapy or after chemotherapy was significantly lower than that before chemotherapy either in the sensitive group or the resistant group (P=0.000). But there was no significant difference between the treatment points during chemotherapy and after chemotherapy in both groups (P values were0.605and0.296). The level of Annexin A3before treatment in the resistant group was significantly higher than that in the sensitive group (P=0.012), and the median concentrations were2.21ng/ml (0.5-6.18ng/ml) and0.80ng/ml (0.20-2.43ng/ml) respectively. But the levels of Annexin A3during or after chemotherapy showed no significant difference between the two groups (P values were0.937and0.624). The level of CA125before treatment showed no significant difference between the two groups (P=0.271) and had no diagnostic value to platinum chemoresistance because the area under ROC curve was only0.597(P=0.271). While the CA125level during chemotherapy in the resistant group was significantly higher than that in the sensitive group (P=0.003) and the AUC was0.755(P=0.014).The best diagnostic cutoff value was24.60U/ml and at this point, the diagnostic sensitivity was60.00%, the specificity was90.24%, the accuracy was82.14%, the false positive rate was9.76%, the false negative rate was40.00%, the positive predictive value was69.23%and the negative predictive value was86.01%. By correlation analysis, the expression level of Annexin A3was unrelated to CA125at any treatment points (P values were0.384,0.594and0.883). Using the upper limit of normal1.45ng/ml as cutoff value, the ovarian cancer patients before treatment was divided into the high-expression group and the low-expression group and the platinum resistance ratio in the high-expression group was significantly higher than that in the low-expression group (55.56%vs13.16%, P=0.001). Logistic regression analysis showed that the level of Annexin A3before treatment was one of the platinum-resistant risk factors (P=0.004, OR=9.241), which meant the platinum resistance risk of high-expression group was9.241times that of the low-expression group. Diagnostic test showed that the AUC of Annexin A3before treatment was0.744(P=0.006) and the best cutoff point was1.645ng/ml. At this point, the sensitivity, specificity, accuracy, false positive rate, false negative rate, positive predictive value and negative predictive value of the prediction to platinum resistance were66.70%,82.93%,78.57%,17.07%,33.30%,58.82%and87.18%respectively. Cox hazard regression model analysis showed that the level of Annexin A3before treatment was an independent risk factor for disease progression (P=0.001, RR=9.863), which meant the risk of disease progression in the high-expression group was9.863times that in the low-expression group. By the Kaplan-Meier survival analysis, the median PFS of the two groups were27.60months (2.23to32.17months) and12.77months (4.90to25.80months) respectively and the PFS of high-expression group was significantly shorter than that of low-expression group (P=0.001).
Conclusions
1、In this study we chose a pair of antibody as the capture antibody and detection antibody from six kinds of commercially available anti-Annexin A3polyclonal or monoclonal antibodies which had the best affinity to human Annexin A3recombinant protein. And the Annexin A3protein chemiluminescence immunoassay kit was successfully prepared for the first time.
2、The CLIA kit had high sensitivity, good reproducibility and dilution linearity, and convenient operation.Compared with currently commercially available ELISA kit, the CLIA kit had higher sensitivity (0.08ng/ml vs0.2ng/ml), better discrimination to serum with low concentration of Annexin A3protein, and fewer steps. The CLIA kit was better than the ELISA kit and could meet the requirements of clinical detection. But the CLIA kit also had a shortcoming of the low upper limit of standard curve, which resulted in the re-test of high value serum samples after dilution. Further improvement was needed in order to be to increase the linear detection range.
3、Annexin A3protein could be detected differential expression in the peripheral blood of different women population by the CLIA kit and its upper limit of normal was initially defined as1.45ng/ml. The level of Annexin A3before treatment in ovarian cancer patients was significantly higher than that in the healthy women or in benign ovarian tumor patients. The level of Annexin A3didn't change after the removal of tumors by surgery but could gradually decreased to normal with chemotherapy and there was no significant difference between the sensitive group and the resistant group. The level of CA125before treatment had no significant difference between the sensitive group and the resistant group, but during chemotherapy the level of CA125in the resistant group was significantly higher than that in sensitive group. The expression level of Annexin A3was unrelated to CA125at any treatment points.The differential expression of Annexin A3before treatment and CA125during chemotherapy between the sensitive and resistant group suggested they had predictive and diagnostic value in judging the platinum chemoresistance respectively. Besides the level of Annexin A3before treatment was the independent risk factor for disease progression.
卵巢癌是死亡率居首位的女性生殖系统恶性肿瘤,肿瘤细胞对化疗药物产生耐药性是临床治疗的主要障碍。运用铂类耐药标记蛋白,早期发现肿瘤耐药,有助于临床及时调整化疗方案,改善患者预后。
本课题组前期运用比较蛋白质组学技术,发现并证实了一种卵巢癌铂类耐药标记蛋白膜联蛋白A3(Annexin A3),该蛋白能够以细胞内囊泡形成、转运、外泌体的形式外泌,在细胞培养液上清和人外周血中均可检测到不同水平的表达,AnnexinA3有望成为一种铂类耐药血清学标记物应用于临床。然而,在扩大样本量进行临床检测的研究中发现,本课题组所应用的目前市场上唯一可提供的ELISA试剂盒存在敏感度低、重复性差等突出问题,造成实验数据的准确性欠佳。因此,研制一种能高通量、稳定检测人外周血Annexin A3蛋白表达水平的免疫分析试剂盒成为本课题深入研究中亟待解决的关键问题。
化学发光免疫分析法(CLIA)是继放射免疫分析法、酶免疫分析法和荧光免疫分析法之后发展起来的一种新的免疫学测定方法,该技术将高特异性的抗原抗体反应与高灵敏度的化学发光反应结合起来,具有灵敏度高、特异性强、重复性好、操作简便、分析快速、无放射污染等优点,成为目前临床检测蛋白分子的主流方法。
本实验在前期有关卵巢癌铂类耐药特异性标记蛋白Annexin A3工作的基础上进行深入研究,旨在制备能够满足临床检测要求的Annexin A3蛋白化学发光试剂盒,并应用该试剂盒对不同女性人群外周血中的Annexin A3蛋白进行检测,以了解该蛋白在人外周血中的表达情况,初步确定其正常值范围,并探讨其作为血清学标志物对临床上卵巢癌患者铂类化疗耐药的预测价值。
研究方法
1、筛选匹配高特异性的Annexin A3抗原及抗体对,制备检测人外周血.中AnnexinA3蛋白的化学发光免疫分析试剂盒。
购买市售的人Annexin A3重组蛋白及六种Annexin A3多克隆、单克隆抗体作为检测抗原和抗体,化学发光免疫分析法检测不同组合的两种抗体与各浓度抗原反应的发光值,绘制标准曲线,计算线性拟合相关系数r及信噪比(最高浓度标准品与0值标准品发光值之比,S5/SO),r值越接近1、信噪比越大表明抗原抗体间的结合特异性越高,选择r值及信噪比最大的两种抗体作为制备试剂盒的捕获抗体和检测抗体。将人Annexin A3重组蛋白稀释成不同浓度作为标准品,将筛选出的捕获抗体包被在固相载体上,采用改良过碘酸钠法连接检测抗体和辣根过氧化物酶制备出酶标检测抗体,两种抗体与抗原形成双抗体夹心复合物,加入发光底物后,使该复合物发光,化学发光仪检测发光强度。交叉方阵滴定法确定捕获抗体与酶标检测抗体的最佳工作浓度,并分别采用一步法和两步法对反应模式进行优化,最终确立最佳检测体系,完成试剂盒的制备。
2、对制备完成的Annexin A3化学发光试剂盒进行灵敏度、精密度和稀释线性的评价,确定检测工作范围,并与本课题组前期研究中应用的目前市场上唯一可提供的Annexin A3酶联免疫吸附法(ELISA)检测试剂盒进行比较评价。
平行测量20个零值标准品(S0),以其发光值(RLU)的平均值(Mean)加上两倍标准差(SD)所对应的浓度值求得本方法的灵敏度。精密度指测定结果的可重复性,评价指标为变异系数(coefficient of variation, CV),分别用1.6ng/mL和0.8ng/mL两种浓度的Annexin A3蛋白作为高、低值质控品,每个质控品平行做20孔,用不同批次的包被板重复测3次,计算单次检测(批内)和多次检测(批间)的CV值,CV(%)=标准差(SD)／均数(Mean)。将1份本课题组前期实验中用ELISA法测定为高值的血清样品(浓度为1.25ng/mL),按1：1、1：2、1：4、1：8、1：16、1：32、1：64进行倍比稀释后测定发光值,对稀释倍数及发光值进行线性拟合,计算相关系数r。将各浓度的标准品重复检测10次,根据每次的标准曲线求得相应浓度值,计算10次测量的均数及标准差,进而求出各浓度点的变异系数CV,以CV为纵坐标,浓度值为横坐标,绘制精密度曲线图,选用图中变异系数小于10%的浓度范围作为试剂盒的工作范围。取健康女性、卵巢良性肿瘤、上皮性卵巢癌患者的血清样本各20份,分别应用两种试剂盒进行检测,比较测定值的分布及对低值血样的区分情况。
3、应用Annexin A3化学发光试剂盒高通量检测人外周血中Annexin A3蛋白的表达水平,确定其正常值范围,评估其作为卵巢癌铂类化疗耐药血清学标志物的可行性,同时与CA125比较在临床诊断卵巢癌耐药中的作用。
连续收集2009年9月至2012年4月期间在北京协和医院体检的健康女性血清、以及住院手术治疗的卵巢良性和交界性肿瘤患者术前血清、上皮性卵巢癌患者治疗前血清,应用CLIA试剂盒检测Annexin A3蛋白在各组人群外周血中的浓度,初步建立其正常参考值范围。前瞻性连续收集卵巢癌患者治疗前、术后化疗前、化疗过程中以及化疗结束后的血清样本,并记录同期的CA125测定值。应用CLIA试剂盒检测Annexin A3的浓度,评估各临床病理因素、手术及化疗对Annexin A3表达水平的影响。对所有卵巢癌患者进行随访,根据目前对卵巢癌铂类耐药的定义判断患者的化疗结局,分为敏感组和耐药组,明确Annexin A3在两组患者不同治疗时点的动态变化及表达差异。与CA125进行相关性分析,探讨各治疗时点的Annexin A3表达水平与铂类耐药的关系,通过诊断试验评估CA125及Annexin A3对铂类耐药的诊断或预测作用：制作受试者工作特征(ROC)曲线,计算曲线下面积(AUC)以及各浓度值对应的约登指数(又称正确诊断指数,等于敏感度与特异度之和减1),AUC=0.5表明无诊断价值,AUC在0.5～0.7时有较低准确性,在0.7～0.9时有一定准确性,在0.9以上有较高准确性,约登指数最大时表明以该对应浓度为界值进行诊断的准确性最高,因此选择该浓度值为最佳诊断界值,计算此时诊断的敏感度、特异度、准确度(总符合率)、假阳性率、假阴性率、阳性预测值及阴性预测值。通过COX风险回归模型及Kaplan-Meier生存分析评估治疗前Annexin A3水平对患者无进展生存期的影响。
研究结果
1、选用兔抗-Annexin A3多克隆抗作为捕获抗体,鼠抗-Annexin A3多克隆抗体作为检测抗体时,与各浓度Annexin A3抗原反应得到的标准曲线的相关系数r及信噪比均最大,分别为0.9982和19.56,故选择两者应用于试剂盒的制备。采用浓度为5μg/mL的捕获抗体包被固相载体,稀释度为1：250的酶标检测抗体进行检测时,与各浓度Annexin A3标准品反应得到的标准曲线的相关系数r及信噪比均最大,分别为0.9987和59.33,从而确定为最佳工作浓度。采用一步法和两步法检测时的相关系数均较好,分别为0.9983和0.9986,但一步法的信噪比略高于两步法(49.11vs45.57),结合操作的简便性,反应模式选择一步法。
2、试剂盒的最低检测浓度即灵敏度为0.08ng/mL;批内精密度为5.92～8.63%,批间精密度为5.15～6.42%；高值血样的稀释度与发光值间呈良好的线性相关,相关系数r为0.9997；标准曲线在0.1～1.6ng/mL浓度之间的变异系数均小于10%,因此选定0.1～1.6ng/mL作为工作范围。两种试剂盒检测结果显示,ELISA试剂盒对60份血样的中位测值为0.36ng/mL (0.1～2.03ng/mL),其中80%(48/60)的血样测值低于标准曲线建议的最低浓度(0.8ng/mL),30%(18/60)低于ELISA试剂盒说明书标明的最低检测浓度(0.2ng/mL); CLIA试剂盒对60份血样的中位测值为0.95ng/mL (0.04～2.77ng/mL),90%(54/60)的血样测值位于标准曲线范围内,其中2例低于试剂盒最低检测浓度(0.08ng/mL),4例高于标准曲线上限(1.6ng/mL),将该4份高值血清分别稀释2倍、4倍、8倍后进行复测,不同稀释倍数得到的最终测值间具有较好的一致性,变异系数均不到3%。CLIA试剂盒的操作步骤也较ELISA试剂盒简单,前者采用样本和酶标检测抗体同时加入的一步法检测模式,反应时间仅需2小时,而后者则需将样本和酶标检测抗体分三步加入,反应时间长达4.5小时。
3、共纳入符合标准的健康女性113例,卵巢良性肿瘤患者70例,卵巢交界性肿瘤患者14例,上皮性卵巢癌患者96例。四组人群外周血Annexin A3的中位浓度分别为0.75ng/mL (0.15～1.54ng/mL)、0.66ng/mL (0.04～1.79ng/mL)、0.65ng/mL (0.14～3.29ng/mL)以及1.12ng/mL (0.20～6.18ng/mL);卵巢癌患者治疗前Annexin A3水平明显高于健康女性及卵巢良性肿瘤患者(P值均为0.000),健康女性与卵巢良性肿瘤患者相比无统计学差异(P=0.322),卵巢交界性肿瘤患者与其余三组人群相比均无统计学差异(P值分别为0.144、0.132、0.236)。Annexin A3在113例健康女性外周血中的表达水平呈近似正态分布(Z=0.723,P=0.673>0.05),采用正态分布法确定参考值范围,正常值上限为Mean+1.96SD=1.45ng/mL
卵巢癌患者治疗前Annexin A3水平与年龄、组织学类型、组织学分级、手术病理分期、淋巴结转移均无关(P值分别为0.985、0.631、0.347、0.441、0.480)；术前及术后化疗前Annexin A3的中位浓度分别为1.11ng/mL (0.43～4.55ng/mL)、1.23ng/mL (0.4～4.32ng/mL),相比无统计学差异(P=0.226)；化疗前、化疗中、化疗后Annexin A3的中位浓度分别为1.15ng/mL (0.42～6.18ng/mL)、0.23ng/mL (0.02～3.00ng/mL)以及0.24ng/mL (0.03～1.82ng/mL),化疗中和化疗后较化疗前明显降低(P值均为0.000),化疗中与化疗后相比无统计学差异(P=0.487)。
截至2012年4月30日随访结束,96例卵巢癌患者的中位随访时间为10.73个月(2.23-32.17个月),共有56例可明确判断铂类耐药性结局,其中铂类敏感患者41例(占73.21%),耐药患者15例(占26.79%)；共有21例患者出现疾病进展,3例失访,3例仍在进行先期化疗,尚未手术。
敏感组患者治疗前、化疗中、化疗后的Annexin A3中位浓度分别0.80ng/mL (0.20～2.43ng/mL).0.31ng/mL(0.02～0.84ng/mL)、0.22ng/mL(0.06～0.75ng/mL),耐药组患者分别为2.21ng/mL (0.5～6.18ng/mL)、0.16ng/mL (0.04～0.76ng/mL)、0.24ng/mL (0.03～1.79ng/mL),两组患者化疗中及化疗后的Annexin A3水平均较化疗前明显降低(P值均为0.000),化疗中与化疗后相比无统计学差异(P值分别为0.605、0.296)；组间比较显示,耐药组患者治疗前Annexin A3水平明显高于敏感组(P=0.012),而化疗中与化疗后两组的Annexin A3水平无明显差别(P值分别为0.937、0.624)。
敏感及耐药患者治疗前CA125的平均浓度分别为(1096.51±1689.91)U/mL、(1896.48±2162.88)U/mL,两组比较无统计学差异(P=0.271),而耐药组患者化疗中CA125的表达水平明显高于敏感组(P=0.003),平均浓度分别为(16.52±12.47)U/mL、(198.89±527.32)U/mL,经相关性分析,治疗前、化疗中及化疗后AnnexinA3的表达水平均与CA125无关(P值分别为0.384、0.594、0.883)。治疗前CA125对铂类耐药无诊断作用,ROC曲线下面积AUC为0.597(P=0.271)；化疗中CA125诊断铂类耐药的AUC为0.755(P=0.014),最佳界值点为24.60U/mL,此时诊断铂类耐药的敏感度为60.00%,特异度为90.24%,准确度为82.14%,假阳性率为40.00%,假阴性率为9.76%,阳性预测值为69.23%,阴性预测值为86.01%。
以正常参考值上限1.45ng/mL为界对治疗前患者进行分组,高水平组发生铂类耐药的比率明显高于低水平组(55.56%vs13.16%,P=0.001),Logistic回归分析显示,治疗前Annexin A3水平是发生铂类耐药的危险因素之一(P=0.004,OR=9.241),高水平组发生铂类耐药的风险是低水平组的9.241倍。诊断试验结果显示,治疗前Annexin A3诊断铂类耐药的AUC为0.744(P=0.006),最佳界值点为1.645ng/mL此时治疗前Annexin A3预测铂类耐药的敏感度为66.70%,特异度为82.93%,准确度为78.57%,假阳性率为17.07%,假阴性率为33.30%,阳性预测值为58.82%,阴性预测值为87.18%。COX风险回归模型分析显示,治疗前Annexin A3水平是影响患者疾病进展的独立危险因素(P=0.001,RR=9.863),高水平组发生疾病进展的风险是低水平组的9.863倍,Kaplan-Meier生存分析显示,两组的中位PFS分别为27.60个月(2.23-32.17个月)和12.77个月(4.90-25.80个月),高水平组的PFS显著短于低水平组(P=0.001)。
结论
1、本研究从市售的六种Annexin A3多克隆、单克隆抗体中筛选出与人Annexin A3重组蛋白特异性结合最好的两种作为制备化学发光试剂盒的捕获抗体和检测抗体,首次成功制备出人Annexin A3蛋白化学发光定量检测试剂盒。
2、本研究制备的Annexin A3化学发光试剂盒具有较高的灵敏度,良好的重复性和稀释线性,操作方便、分析迅速,明显优于市售ELISA试剂盒,能够满足临床检测的要求,但该试剂盒还存在检测上限值偏低、高值血清样本需重新稀释后复测的缺点,需进一步改善以增加检测的线性范围。
3、应用本研究研制的化学发光试剂盒可以检测到不同人群外周血中Annexin A3蛋白的差异表达,卵巢癌患者治疗前Annexin A3水平明显高于健康女性及卵巢良性肿瘤患者,初步确定女性外周血Annexin A3的正常参考值上限。前瞻性观察56例可随诊结局的卵巢上皮癌患者,治疗前耐药组Annexin A3的的测定值明显高于敏感组,化疗期间耐药组CA125的的测定值明显高于敏感组,提示卵巢癌患者治疗前外周血Annexin A3的测定值对于临床出现铂类耐药具有一定的预测作用,化疗期间CA125测定值对于临床出现铂类耐药具有一定的诊断作用。此外,治疗前Annexin A3水平还是影响患者疾病进展的独立危险因素,是判断预后的重要指标。
Backgroud and Purpose
Ovarian cancer is the most lethal gynecological malignancy. Resistance of tumor cells to chemotherapeutic drugs is one of the main causes of treatment failure. Using a biomarker to predict clinical platinum resistance is important to treat patients individually and eventually improve prognosis.
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 also demonstrated that Annexin A3was a secreted protein. It could be secreted outside the cells by exosomes and be detected both in culture medium supernatant of ovarian cancer cell lines and serum of ovarian cancer patients. This characteristic enabled Annexin A3as a potential serological platinum resistant biomarker used in clinic. In order to explore the feasibility of quantitative detection of Annexin A3protein in the serum, our research team used the only commercially available detection kit—Annexin A3enzyme-linked immunosorbent assay (ELISA) kit. However, this kit was found the disadvantages such as low sensitivity, poor reproducibility and so on in subsequent clinical detection of large sample size. These outstanding problems resulted in poor accuracy of experimental data. Therefore, developing a new immunoassay kit for high-throughput and stably detecting the expression level of Annexin A3protein in human peripheral blood has become a key issue to be solved in our further study.
Chemiluminescence immunoassay (CLIA) is a new immunological detection method, which develops following radioimmunoassay, enzyme immunoassay and fluorescence immunoassay. It combines highly specific antigen-antibody reaction with high sensitivity chemiluminescent reaction, and has many advantages such as high sensitivity and specificity, good reproducibility, simple and convenient operation, without radioactive contamination and so on. CLIA has become the mainstream method in clinical detection of protein molecule.
Based on above background, the present study aimed to prepare the Annexin A3CLIA kit which can reach clinical detection requirements, and then applied it to quantitatively detect serum Annexin A3protein in different women population in order to understand its expression distribution in human peripheral blood, determine its normal range initially, and explore its predictive value for platinum chemoresistance in ovarian cancer patients as a serological marker.
Methods
1、Six kinds of commercially available anti-Annexin A3polyclonal or monoclonal antibodies were evaluated for their affinity with the antigen—human Annexin A3recombinant protein using double-antibody sandwich chemical luminescence enzyme immunoassay. The best pair was chosen as the capture antibody and detection antibody to prepare chemiluminescence immunoassay kit. Capture antibody was coated on microplate strips in different concentrations and detection antibody was conjugated with horseradish peroxidase (HRP) with modified sodium iodide method which was also diluted in different concentrations. They could form a double-antibody sandwich complex with Annexin A3antigen standard of different concentrations. Luminescence analyzer could detect the light intensity after adding light-emitting substrate which was proportional to the concentration of standard Annexin A3protein. The optimal working concentration of capture antibody and HRP labeled detection antibody was determined with cross-titration method for the best accuracy in measuring known concentrations of antigen. After optimizing the response mode, the final optimal detection system was established and the CLIA kit was prepared successfully.
2、Assess the Annexin A3CLIA kit in its sensitivity, precision (also called reproducibility) and dilution linearity, and determine the detection range by plotting the precision curve. The CLIA kit was also compared with the purchased ELISA kit by simultaneously detecting Annexin A3protein in sixty serum samples from healthy women and patients with benign ovarian tumor or epithelial ovarian cancer.
3、The serum specimens of healthy women at health checkup, benign or borderline ovarian tumor patients before surgery and epithelial ovarian cancer patients before treatment were collected continuously in Peking Union Medical College Hospital from September2009to April2012, and were detected by CLIA kits. The normal reference range of Annexin A3in healthy women was determined by95%confidence interval. The serum specimens of epithelial ovarian cancer patients before surgery or chemotherapy, after surgery but before chemotherapy, during chemotherapy and after chemotherapy were collected prospectively and continuously, all of which were detected by CLIA kits too. The relationship between the concentration of Annexin A3and the various clinicopathologic factors, surgery or chemotherapy was analyzed. All of the ovarian cancer patients were followed up in order to determine the chemotherapy outcome and disease status. According to the definition of clinical platinum chemoresistance, they were divided into the sensitive group and resistant group. The dynamic changes and expression differences of Annexin A3were analyzed in the two groups. By correlation analysis, the relationship between Annexin A3and CA125at different treatment points was evaluated. Diagnostic test was used to assess the diagnostic or predictive value of CA125and Annexin A3.Receiver operating characteristic (ROC) curve was drawn and the best diagnostic cutoff value was determined by the maximum Youden index which is equal to the sensitivity and specificity minus1, and then the diagnostic sensitivity, specificity, accuracy (total coincidence rate), false positive rate, the false negative rate, positive predictive value and negative predictive value were calculated. The ovarian cancer patients were also divided into the high-expression group and the low-expression group using the upper limit of normal for Annexin A3as cutoff value. The correlation between the Annexin A3level before treatment and progression free survival (PFS) was assessed by COX regression analysis and survival analysis.
Results
1、Using a rabbit anti-Annexin A3polyclonal antibody as capture antibody and a mouse anti-Annexin A3polyclonal antibody as detecting antibody to detect Annexin A3standard of different concentration showed the highest antigen-antibody binding specificity based on the largest correlation coefficient of the standard curve and the highest signal to noise ratio (SNR, the light intensity ratio of maximum value and zero value standard proteins),0.9982and19.56respectively. So this antibody pair was the best for preparing CLIA kit. The optimal working concentration of capture antibody and HRP labeled detection antibody was5μg/mL and1:250respectively which was also determined by the largest correlation coefficient0.9987and the highest SNR59.33. The reaction mode of one-step was better than two-step because of the similar correlation coefficient (0.9983vs0.9986) but higher SNR (49.11vs45.57) and easier to operate.
2、The sensitivity of Annexin A3CLIA kit was0.08ng/ml. The intra-assay precision was5.92～8.63%and the inter-assay precision was5.15～6.42%. It had good dilution linearity and the correlation coefficient of the fitting curve between sample dilution and light intensity was0.9997.The work scope of the kit was determined as0.1～1.6ng/ml for the coefficient of variation all less than10%. Detecting sixty blood samples showed the results as follows. The median measured value was0.36ng/ml (0.1～2.03ng/ml) by ELISA kit and0.95ng/ml (0.04～2.77ng/ml) by CLIA kit.80%(48/60) measured value were lower than the minimum recommended concentration (0.8ng/ml) of the standard curve, and30%(18/60) were lower than the minimum detectable concentration (0.2ng/mL) indicate by ELISA kit instructions. While using CLIA kit,90%(54/60) were in the range of the standard curve, only two lower than the minimum detectable concentration (0.08ng/ml). And the other four cases higher than the upper limit of the standard curve (1.6ng/ml) were re-tested by diluted two times, four times and eight times. It showed a good agreement among the final calculated values of different dilutions, and the coefficients of variation were all less than3%. The standard or samples were added at the same time in a one-step detection mode and the reaction time was only two hours. While the ELISA kit applied three-step mode by adding the standard or samples first, then detection antibody and last enzyme which made the reaction time up to4.5hours.
3、113healthy women,70patients with benign ovarian tumor,14patients with borderline ovarian tumor and96patients with epithelial ovarian cancer were enrolled into the study. The median concentration of serum Annexin A3in the four groups was0.75ng/ml (0.15～1.54ng/ml),0.66ng/ml (0.04～1.79ng/ml),0.65ng/ml (0.14～3.29ng/ml) and1.12ng/ml (0.20～6.18ng/ml) respectively. The concentration of Annexin A3before treatment in ovarian cancer patients was significantly higher than that in healthy women and patients with benign ovarian tumor (P=0.000). There was no significant difference between healthy women and patients with benign ovarian tumors (P=0.322). The level of Annexin A3had no significant difference in patients with borderline ovarian tumor compared with the other three groups respectively (P values were0.144,0.132and0.236). Kolmogorov-Smirnov test showed normal distribution of Annexin A3in healthy women (Z=0.723, P=0.673). The upper limit of normal for Annexin A3was defined as1.45ng/ml based on the mean value plus1.96standard deviations.
There was no correlation between the Annexin A3level before treatment and the age, histological type, histological grade, clinical stage, or lymph node metastasis in ovarian cancer patients (P values were0.985,0.631,0.347,0.441and0.480). The median concentrations of Annexin A3before and after surgery were1.11ng/ml (0.43～4.55ng/ml) and1.23ng/ml (0.4～4.32ng/ml) respectively, and there was no significant difference by comparison (P=0.226). The median concentration of Annexin A3before chemotherapy was1.15ng/ml (0.42～6.18ng/ml) and significantly reduced to0.23ng/ml (0.02～3.OOng/ml) during chemotherapy (P=0.000). After chemotherapy, the median concentration of Annexin A3was0.24ng/ml(0.03～1.82ng/ml), significantly lower than that before chemotherapy (P=0.000) but without significant difference compared with that during chemotherapy (P=0.487).
Up to April30,2012, the median follow-up time was10.73months (2.23to32.17months) in96patients with ovarian cancer, of whom56cases could be clearly judged the outcome of platinum chemoresistance, including41(73.21%) platinum-sensitive patients and15(26.79%) drug-resistant patients. During the follow-up period,3patients were lost,21patients had progressive disease, and3patients were still undergoing the neoadjuvant chemotherapy. The level of Annexin A3during chemotherapy or after chemotherapy was significantly lower than that before chemotherapy either in the sensitive group or the resistant group (P=0.000). But there was no significant difference between the treatment points during chemotherapy and after chemotherapy in both groups (P values were0.605and0.296). The level of Annexin A3before treatment in the resistant group was significantly higher than that in the sensitive group (P=0.012), and the median concentrations were2.21ng/ml (0.5-6.18ng/ml) and0.80ng/ml (0.20-2.43ng/ml) respectively. But the levels of Annexin A3during or after chemotherapy showed no significant difference between the two groups (P values were0.937and0.624). The level of CA125before treatment showed no significant difference between the two groups (P=0.271) and had no diagnostic value to platinum chemoresistance because the area under ROC curve was only0.597(P=0.271). While the CA125level during chemotherapy in the resistant group was significantly higher than that in the sensitive group (P=0.003) and the AUC was0.755(P=0.014).The best diagnostic cutoff value was24.60U/ml and at this point, the diagnostic sensitivity was60.00%, the specificity was90.24%, the accuracy was82.14%, the false positive rate was9.76%, the false negative rate was40.00%, the positive predictive value was69.23%and the negative predictive value was86.01%. By correlation analysis, the expression level of Annexin A3was unrelated to CA125at any treatment points (P values were0.384,0.594and0.883). Using the upper limit of normal1.45ng/ml as cutoff value, the ovarian cancer patients before treatment was divided into the high-expression group and the low-expression group and the platinum resistance ratio in the high-expression group was significantly higher than that in the low-expression group (55.56%vs13.16%, P=0.001). Logistic regression analysis showed that the level of Annexin A3before treatment was one of the platinum-resistant risk factors (P=0.004, OR=9.241), which meant the platinum resistance risk of high-expression group was9.241times that of the low-expression group. Diagnostic test showed that the AUC of Annexin A3before treatment was0.744(P=0.006) and the best cutoff point was1.645ng/ml. At this point, the sensitivity, specificity, accuracy, false positive rate, false negative rate, positive predictive value and negative predictive value of the prediction to platinum resistance were66.70%,82.93%,78.57%,17.07%,33.30%,58.82%and87.18%respectively. Cox hazard regression model analysis showed that the level of Annexin A3before treatment was an independent risk factor for disease progression (P=0.001, RR=9.863), which meant the risk of disease progression in the high-expression group was9.863times that in the low-expression group. By the Kaplan-Meier survival analysis, the median PFS of the two groups were27.60months (2.23to32.17months) and12.77months (4.90to25.80months) respectively and the PFS of high-expression group was significantly shorter than that of low-expression group (P=0.001).
Conclusions
1、In this study we chose a pair of antibody as the capture antibody and detection antibody from six kinds of commercially available anti-Annexin A3polyclonal or monoclonal antibodies which had the best affinity to human Annexin A3recombinant protein. And the Annexin A3protein chemiluminescence immunoassay kit was successfully prepared for the first time.
2、The CLIA kit had high sensitivity, good reproducibility and dilution linearity, and convenient operation.Compared with currently commercially available ELISA kit, the CLIA kit had higher sensitivity (0.08ng/ml vs0.2ng/ml), better discrimination to serum with low concentration of Annexin A3protein, and fewer steps. The CLIA kit was better than the ELISA kit and could meet the requirements of clinical detection. But the CLIA kit also had a shortcoming of the low upper limit of standard curve, which resulted in the re-test of high value serum samples after dilution. Further improvement was needed in order to be to increase the linear detection range.
3、Annexin A3protein could be detected differential expression in the peripheral blood of different women population by the CLIA kit and its upper limit of normal was initially defined as1.45ng/ml. The level of Annexin A3before treatment in ovarian cancer patients was significantly higher than that in the healthy women or in benign ovarian tumor patients. The level of Annexin A3didn't change after the removal of tumors by surgery but could gradually decreased to normal with chemotherapy and there was no significant difference between the sensitive group and the resistant group. The level of CA125before treatment had no significant difference between the sensitive group and the resistant group, but during chemotherapy the level of CA125in the resistant group was significantly higher than that in sensitive group. The expression level of Annexin A3was unrelated to CA125at any treatment points.The differential expression of Annexin A3before treatment and CA125during chemotherapy between the sensitive and resistant group suggested they had predictive and diagnostic value in judging the platinum chemoresistance respectively. Besides the level of Annexin A3before treatment was the independent risk factor for disease progression.
引文
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[18]王克珍,买制刚,蒋宏伟等.新型癌抗原125化学发光免疫试剂盒的制备[J].中国生物制品学杂志.2008,21(12)：1105-1107.
[19]李振甲,应希堂,马世俊.化学发光免疫分析技术的研究现状与展望[J].国际检验医学杂志,2006,27(1),95-97.
[20]陈彬,李丽萍,李克.如何确定参考值的范围[J].中华预防医学杂志,2002,36(5)：355-357.
[21]Hennessy BT, Coleman RL, Markman M. Ovarian cancer[J]. Lancet. 2009;17;374(9698):1371-1382.
[22]Goff B, Holmberg L, Veljovich D, Kurland B. Treatment of recurrent or persistent platinum-refractory ovarian, fallopian tube or primary peritoneal cancer with gemcitabine and topotecanA phase Ⅱ trial of the Puget Sound Oncology Consortium. Gynecol Oncol[J].2008;110(2):146-151.
[23]Fu S, HuW, Iyer R, et al. Phase lb-2a study to reverse platinum resistance through use of a hypomethylating agent, azacitidine, in patients with platinum-resistant or platinum-refractory epithelial ovarian cancer[J]. Cancer.2011;117:1661-1669.
[24]Katsumata N, Yasuda M, Takahashi F, Isonishi S, Jobo T, Aoki D, et al. Dose-dense paclitaxel once a week in combination with carboplatin every 3 weeks for advanced ovarian cancer:a phase 3, open-label, randomised controlled trial[J]. Lancet.2009;17;374(9698):1331-1338.
[25]Edge SB, Byrd DR, Compton CC, et al, eds. AJCC Cancer Staging Manual,7th ed. New York:Springer;2010.
[26]Schostak M, Schwall GP, Poznanovic S, Groebe K, Muller M, Messinger D, et al. Annexin A3 in urine:a highly specific noninvasive marker for prostate cancer early detection[J]. J Urol.2009;181(1):343-353.
[1]Gerke V, Moss SE. Annexins:from structure to function[J]. Physiol Rev. 2002;82(2):331-371.
[2]Moss SE, Morgan RO. The annexins[J]. Genome Biol.2004;5(4):219.
[3]Hayes MJ, Moss SE. Annexins and disease[J]. Biochem Biophys Res Commun. 2004;322(4):1166-1170.
[4]Fatimathas L, Moss SE.Annexins as disease modifiers[J]. Histol Histopathol. 2010;25(4):527-32.
[5]Mussunoor S, Murray GI. The role of annexins in tumour development and progression[J]. J Pathol.2008;216(2):131-40.
[6]Tait JF, Frankenberry DA, Miao CH, Killary AM, Adler DA, Disteche CM. Chromosomal localization of the human annexin Ⅲ (ANX3) gene[J]. Genomics.1991;10(2):441-448.
[7]Tait JF, Smith C, Xu L, Cookson BT. Structure and polymorphisms of the humanannexin Ⅲ (ANX3) gene[J]. Genomics.1993;18(1):79-86.
[8]Markoff A, Gerke V. Expression and functions of annexins in the kidney[J]. Am J Physiol Renal Physiol.2005;289(5):949-956.
[9]Hofmann A, Raguenes-Nicol C, Favier-Perron B, Mesonero J, Huber R, Russo-Marie F, et al. The annexin A3-membrane interaction is modulated by an N-terminal tryptophan[J]. Biochemistry.2000;39(26):7712-21.
[10]Eskesen K, Kristensen BI, et al. Calcium-dependent association of annexins with lipid bilayers modifies gramicidin A channel parameters [J]. Eur Biophys J. 2001;30(1):27-33.
[11]Tait JF, Sakata M, McMullen BA, Miao CH, Funakoshi T, Hendrickson LE, et al. Placental anticoagulant proteins:isolation and comparative characterization four members of the lipocortin family[J]. Biochemistry.1988;27(17):6268-6276.
[12]Sopkova J, Raguenes-Nicol C, Vincent M, Chevalier A, Lewit-Bentley A, Russo-Marie F, et al. Ca(2+) and membrane binding to annexin 3 modulate the structure and dynamics of its N terminus and domain Ⅲ [J]. Protein Sci.2002;11(7):1613-1625.
[13]Niimi S, Harashima M, Gamou M, Hyuga M, Seki T, Ariga T, et al. Expression of annexin A3 in primary cultured parenchymal rat hepatocytes and inhibition of DNA synthesis by suppression of annexin A3 expression using RNA interference[J]. Biol Pharm Bull.2005;28(3):424-428.
[14]Harashima M, Harada K, Ito Y, Hyuga M, Seki T, Ariga T, et al. Annexin A3 expression increases in hepatocytes and is regulated by hepatocyte growth factor in rat liver regeneration[J]. J Biochem.2008;143(4):537-545.
[15]Harashima M, Niimi S, Koyanagi H, Hyuga M, Noma S, Seki T, Ariga T, Kawanishi T, Hayakawa T. Change in annexin A3 expression by regulatory factors of hepatocyte growth in primary cultured rat hepatocytes[J]. Biol Pharm Bull.2006;29(7):1339.
[16]Konishi H, Namikawa K, Kiyama H. Annexin Ⅲ implicated in the microglial response to motor nerve injury[J]. Glia.2006;53(7):723-32.
[17]Park JE, Lee DH, Lee JA, Park SG, Kim NS, Park BC, et al. Annexin A3 is a potential angiogenic mediator[J]. Biochem Biophys Res Commun.2005;337(4):1283-7.
[18]Wozny W, Schroer K, Schwall GP, Poznanovic S, Stegmann W, Dietz K, et al. Differential radioactive quantification of protein abundance ratios between benign and malignant prostate tissues:cancer association of annexin A3[J]. Proteomics.2007;7(2):313-22.
[19]Kollermann J, Schlomm T, Bang H, Schwall GP, von Eichel-Streiber C, Simon R, et al. Expression and prognostic relevance of annexin A3 in prostate cancer[J]. Eur Urol.2008;54(6):1314-23.
[20]Pisitkun T, Shen RF, Knepper MA. Identification and proteomic profiling of exosomes in human urine[J]. Proc Natl Acad Sci USA.2004;101:13368-13373.
[21]Schostak M, Schwall GP, et al. Annexin A3 in urine:a highly specific noninvasive marker for prostate cancer early detection[J]. J Urol.2009;181(1):343-353.
[22]Cao DL, Ye DW, Zhang HL, Zhu Y, Wang YX, Yao XD. A multiplex model of combining gene-based, protein-based, and metabolite-based with positive and negative markers in urine for the early diagnosis of prostate cancer[J]. Prostate. 2011;71(7):700-710.
[23]Thoenes L, Hoehn M, Kashirin R, Ogris M, Arnold GJ, Wagner E, Guenther M. In vivo chemoresistance of prostate cancer in metronomic cyclophosphamide therapy[J]. J Proteomics.2010;73(7):1342-54.
[24]Jung EJ, Moon HG, Park ST, Cho BI, Lee SM, Jeong CY, et al. Decreased annexin A3 expression correlates with tumor progression in papillary thyroid cancer[J]. Proteomics Clin Appl.2010;4(5):528-537.
[25]Bianchi C, Bombelli S, Raimondo F, Torsello B, Angeloni V, Ferrero S, et al. Primary cell cultures from human renal cortex and renal-cell carcinoma evidence a differential expression of two spliced isoforms of Annexin A3 [J]. Am J Pathol.2010;176(4):1660-1670.
[26]Yan XD, Li M, Yuan Y, Mao M, Pan LY. Biological comparison of ovarian cancer resistant cell lines to cisplatin and Taxol by two different administrations[J]. Oncol Rep.2007;17(5):1163-69.
[27]Yan XD, Pan LY, Yuan Y, Lang JH, Mao N. Identification of platinum-resistance associated proteins through proteomic analysis of human ovarian cancer cells and their platinum-resistant sublines[J]. J Proteome Res.2007;6:772-80.
[28]Yan X, Yin J, Yao H, Mao N, Yang Y, and Pan L. Increased expression of annexin A3 is a mechanism of platinum resistance in ovarian cancer[J]. Cancer Res.2010;70:1616-1624.
[29]Yin J, Yan X, Yao X, Zhang Y, Shan Y, Mao N, et al. Secretion of annexin A3 from ovarian cancer cells and its association with platinum resistance in ovarian cancer patients[J]. J Cell Mol Med.2012;16(2):337-348.
[30]Madoz-Gurpide J, Lopez-Serra P, Martinez-Torrecuadrada JL, Sanchez L, Lombardia L, Casal JI.Proteomics-based validation of genomic data:applications in colorectal cancer diagnosis[J]. Molec Cell Proteomics.2006;5(8):1471-1483.
[31]Marshall KW, Mohr S, Khettabi FE et al. A blood-based biomarker panel for stratifying current risk for colorectal cancer[J]. Int J Cancer.2010;126(5):1177-86.
[32]Yip KT, Das PK, Suria D, Lim CR, Ng GH, Liew CC. A case-controlled validation study of a blood based seven gene biomarker panel for colorectal cancer in Malaysia[J].J Exp Clin Cancer Res.2010;29:128.
[33]Liu YF, Xiao ZQ, Li MX, Li MY, Zhang PF, Li C, et al. Quantitative proteome analysis reveals annexin A3 as a novel biomarker in lung adenocarcinoma[J]. J Pathol.2009;217(1):54-64.
[34]Wang JW, Peng SY, Li JT, Wang Y, Zhang ZP, Cheng Y, et al. Identification of metastasis-associated proteins involved in gallbladder carcinoma metastasis by proteomic analysis and functional exploration of chloride intracellular channel 1[J]. Cancer Lett.2009;281(1):71-81.
[35]谈燚,孟海萍,吴穷,王富强,吴浩荣.胆囊癌组织蛋白质组研究及膜联蛋白A3表达的临床病理意义[J].中华病理学杂志,2010,39(6)：382-386.
[36]Baine MJ, Chakraborty S, Smith LM, Mallya K, Sasson AR, Brand RE, Batra SK. Transcriptional profiling of peripheral blood mononuclear cells in pancreatic cancer patients identifies novel genes with potential diagnostic utility[J]. Plos One. 2011;6(2):e17014.
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[6]Yan XD, Pan LY, Yuan Y, Lang JH, Mao N. Identification of Platinum-Resistance Associated Proteins through Proteomic Analysis of Human Ovarian Cancer Cells and Their Platinum-Resistant Sublines[J]. J Proteome Res.2007;6:772-80.
[7]Yan X, Yin J, Yao H, Mao N, Yang Y, and Pan L. Increased Expression of Annexin A3 Is a Mechanism of Platinum Resistance in Ovarian Cancer[J]. Cancer Res.2010;70:1616-1624.
[8]Yin J, Yan X, Yao X, Zhang Y, Shan Y, Mao N, et al. Secretion of annexin A3 from ovarian cancer cells and its association with platinum resistance in ovarian cancer patients[J]. J Cell Mol Med.2012;16(2):337-348.
[9]Safi MA. An overview of various labeled assays used in medical laboratory diagnosis[J]. Immune and non-immune assays. Saudi Med J.2010;31(4):359-368.
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[16]Rilo I, Takashi H. Direct determination of plasma endothelin-II by chemil uminescence enzyme immunoassay[J]. Clin Chem,1996;42(8):1155-1158.
[17]Salles B, Provot C. In vitro chemiluminescence assay to measure excision repair in cell extracts[J]. Methods Mol Biol.1999;113:393-401.
[18]王克珍,买制刚,蒋宏伟等.新型癌抗原125化学发光免疫试剂盒的制备[J].中国生物制品学杂志.2008,21(12)：1105-1107.
[19]李振甲,应希堂,马世俊.化学发光免疫分析技术的研究现状与展望[J].国际检验医学杂志,2006,27(1),95-97.
[20]陈彬,李丽萍,李克.如何确定参考值的范围[J].中华预防医学杂志,2002,36(5)：355-357.
[21]Hennessy BT, Coleman RL, Markman M. Ovarian cancer[J]. Lancet. 2009;17;374(9698):1371-1382.
[22]Goff B, Holmberg L, Veljovich D, Kurland B. Treatment of recurrent or persistent platinum-refractory ovarian, fallopian tube or primary peritoneal cancer with gemcitabine and topotecanA phase Ⅱ trial of the Puget Sound Oncology Consortium. Gynecol Oncol[J].2008;110(2):146-151.
[23]Fu S, HuW, Iyer R, et al. Phase lb-2a study to reverse platinum resistance through use of a hypomethylating agent, azacitidine, in patients with platinum-resistant or platinum-refractory epithelial ovarian cancer[J]. Cancer.2011;117:1661-1669.
[24]Katsumata N, Yasuda M, Takahashi F, Isonishi S, Jobo T, Aoki D, et al. Dose-dense paclitaxel once a week in combination with carboplatin every 3 weeks for advanced ovarian cancer:a phase 3, open-label, randomised controlled trial[J]. Lancet.2009;17;374(9698):1331-1338.
[25]Edge SB, Byrd DR, Compton CC, et al, eds. AJCC Cancer Staging Manual,7th ed. New York:Springer;2010.
[26]Schostak M, Schwall GP, Poznanovic S, Groebe K, Muller M, Messinger D, et al. Annexin A3 in urine:a highly specific noninvasive marker for prostate cancer early detection[J]. J Urol.2009;181(1):343-353.
[1]Gerke V, Moss SE. Annexins:from structure to function[J]. Physiol Rev. 2002;82(2):331-371.
[2]Moss SE, Morgan RO. The annexins[J]. Genome Biol.2004;5(4):219.
[3]Hayes MJ, Moss SE. Annexins and disease[J]. Biochem Biophys Res Commun. 2004;322(4):1166-1170.
[4]Fatimathas L, Moss SE.Annexins as disease modifiers[J]. Histol Histopathol. 2010;25(4):527-32.
[5]Mussunoor S, Murray GI. The role of annexins in tumour development and progression[J]. J Pathol.2008;216(2):131-40.
[6]Tait JF, Frankenberry DA, Miao CH, Killary AM, Adler DA, Disteche CM. Chromosomal localization of the human annexin Ⅲ (ANX3) gene[J]. Genomics.1991;10(2):441-448.
[7]Tait JF, Smith C, Xu L, Cookson BT. Structure and polymorphisms of the humanannexin Ⅲ (ANX3) gene[J]. Genomics.1993;18(1):79-86.
[8]Markoff A, Gerke V. Expression and functions of annexins in the kidney[J]. Am J Physiol Renal Physiol.2005;289(5):949-956.
[9]Hofmann A, Raguenes-Nicol C, Favier-Perron B, Mesonero J, Huber R, Russo-Marie F, et al. The annexin A3-membrane interaction is modulated by an N-terminal tryptophan[J]. Biochemistry.2000;39(26):7712-21.
[10]Eskesen K, Kristensen BI, et al. Calcium-dependent association of annexins with lipid bilayers modifies gramicidin A channel parameters [J]. Eur Biophys J. 2001;30(1):27-33.
[11]Tait JF, Sakata M, McMullen BA, Miao CH, Funakoshi T, Hendrickson LE, et al. Placental anticoagulant proteins:isolation and comparative characterization four members of the lipocortin family[J]. Biochemistry.1988;27(17):6268-6276.
[12]Sopkova J, Raguenes-Nicol C, Vincent M, Chevalier A, Lewit-Bentley A, Russo-Marie F, et al. Ca(2+) and membrane binding to annexin 3 modulate the structure and dynamics of its N terminus and domain Ⅲ [J]. Protein Sci.2002;11(7):1613-1625.
[13]Niimi S, Harashima M, Gamou M, Hyuga M, Seki T, Ariga T, et al. Expression of annexin A3 in primary cultured parenchymal rat hepatocytes and inhibition of DNA synthesis by suppression of annexin A3 expression using RNA interference[J]. Biol Pharm Bull.2005;28(3):424-428.
[14]Harashima M, Harada K, Ito Y, Hyuga M, Seki T, Ariga T, et al. Annexin A3 expression increases in hepatocytes and is regulated by hepatocyte growth factor in rat liver regeneration[J]. J Biochem.2008;143(4):537-545.
[15]Harashima M, Niimi S, Koyanagi H, Hyuga M, Noma S, Seki T, Ariga T, Kawanishi T, Hayakawa T. Change in annexin A3 expression by regulatory factors of hepatocyte growth in primary cultured rat hepatocytes[J]. Biol Pharm Bull.2006;29(7):1339.
[16]Konishi H, Namikawa K, Kiyama H. Annexin Ⅲ implicated in the microglial response to motor nerve injury[J]. Glia.2006;53(7):723-32.
[17]Park JE, Lee DH, Lee JA, Park SG, Kim NS, Park BC, et al. Annexin A3 is a potential angiogenic mediator[J]. Biochem Biophys Res Commun.2005;337(4):1283-7.
[18]Wozny W, Schroer K, Schwall GP, Poznanovic S, Stegmann W, Dietz K, et al. Differential radioactive quantification of protein abundance ratios between benign and malignant prostate tissues:cancer association of annexin A3[J]. Proteomics.2007;7(2):313-22.
[19]Kollermann J, Schlomm T, Bang H, Schwall GP, von Eichel-Streiber C, Simon R, et al. Expression and prognostic relevance of annexin A3 in prostate cancer[J]. Eur Urol.2008;54(6):1314-23.
[20]Pisitkun T, Shen RF, Knepper MA. Identification and proteomic profiling of exosomes in human urine[J]. Proc Natl Acad Sci USA.2004;101:13368-13373.
[21]Schostak M, Schwall GP, et al. Annexin A3 in urine:a highly specific noninvasive marker for prostate cancer early detection[J]. J Urol.2009;181(1):343-353.
[22]Cao DL, Ye DW, Zhang HL, Zhu Y, Wang YX, Yao XD. A multiplex model of combining gene-based, protein-based, and metabolite-based with positive and negative markers in urine for the early diagnosis of prostate cancer[J]. Prostate. 2011;71(7):700-710.
[23]Thoenes L, Hoehn M, Kashirin R, Ogris M, Arnold GJ, Wagner E, Guenther M. In vivo chemoresistance of prostate cancer in metronomic cyclophosphamide therapy[J]. J Proteomics.2010;73(7):1342-54.
[24]Jung EJ, Moon HG, Park ST, Cho BI, Lee SM, Jeong CY, et al. Decreased annexin A3 expression correlates with tumor progression in papillary thyroid cancer[J]. Proteomics Clin Appl.2010;4(5):528-537.
[25]Bianchi C, Bombelli S, Raimondo F, Torsello B, Angeloni V, Ferrero S, et al. Primary cell cultures from human renal cortex and renal-cell carcinoma evidence a differential expression of two spliced isoforms of Annexin A3 [J]. Am J Pathol.2010;176(4):1660-1670.
[26]Yan XD, Li M, Yuan Y, Mao M, Pan LY. Biological comparison of ovarian cancer resistant cell lines to cisplatin and Taxol by two different administrations[J]. Oncol Rep.2007;17(5):1163-69.
[27]Yan XD, Pan LY, Yuan Y, Lang JH, Mao N. Identification of platinum-resistance associated proteins through proteomic analysis of human ovarian cancer cells and their platinum-resistant sublines[J]. J Proteome Res.2007;6:772-80.
[28]Yan X, Yin J, Yao H, Mao N, Yang Y, and Pan L. Increased expression of annexin A3 is a mechanism of platinum resistance in ovarian cancer[J]. Cancer Res.2010;70:1616-1624.
[29]Yin J, Yan X, Yao X, Zhang Y, Shan Y, Mao N, et al. Secretion of annexin A3 from ovarian cancer cells and its association with platinum resistance in ovarian cancer patients[J]. J Cell Mol Med.2012;16(2):337-348.
[30]Madoz-Gurpide J, Lopez-Serra P, Martinez-Torrecuadrada JL, Sanchez L, Lombardia L, Casal JI.Proteomics-based validation of genomic data:applications in colorectal cancer diagnosis[J]. Molec Cell Proteomics.2006;5(8):1471-1483.
[31]Marshall KW, Mohr S, Khettabi FE et al. A blood-based biomarker panel for stratifying current risk for colorectal cancer[J]. Int J Cancer.2010;126(5):1177-86.
[32]Yip KT, Das PK, Suria D, Lim CR, Ng GH, Liew CC. A case-controlled validation study of a blood based seven gene biomarker panel for colorectal cancer in Malaysia[J].J Exp Clin Cancer Res.2010;29:128.
[33]Liu YF, Xiao ZQ, Li MX, Li MY, Zhang PF, Li C, et al. Quantitative proteome analysis reveals annexin A3 as a novel biomarker in lung adenocarcinoma[J]. J Pathol.2009;217(1):54-64.
[34]Wang JW, Peng SY, Li JT, Wang Y, Zhang ZP, Cheng Y, et al. Identification of metastasis-associated proteins involved in gallbladder carcinoma metastasis by proteomic analysis and functional exploration of chloride intracellular channel 1[J]. Cancer Lett.2009;281(1):71-81.
[35]谈燚,孟海萍,吴穷,王富强,吴浩荣.胆囊癌组织蛋白质组研究及膜联蛋白A3表达的临床病理意义[J].中华病理学杂志,2010,39(6)：382-386.
[36]Baine MJ, Chakraborty S, Smith LM, Mallya K, Sasson AR, Brand RE, Batra SK. Transcriptional profiling of peripheral blood mononuclear cells in pancreatic cancer patients identifies novel genes with potential diagnostic utility[J]. Plos One. 2011;6(2):e17014.