卵巢癌铂类耐药标记蛋白Annexin A3对临床化疗耐药预测作用的前瞻性研究
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摘要
研究背景及目的
卵巢上皮癌是女性生殖器官中死亡率居第一位的恶性肿瘤,目前的标准治疗方案是在肿瘤细胞减灭术的基础上辅以紫杉醇和铂类联合的一线化疗,但是预后不理想,晚期患者的5年生存率仍然徘徊在30%左右。肿瘤细胞对于化疗药物产生耐药性是造成卵巢癌的5年生存率得不到进一步改善的主要原因之一,初治患者的完全缓解率接近75%,仍有15-30%的患者对初治方案反应不佳；在达到完全缓解的患者中,有>90%的患者肿瘤复发。如何早期发现和诊断化疗耐药,进一步改善卵巢癌的预后是当前困扰妇科肿瘤临床的主要问题之一。卵巢癌患者对铂类药物耐药的临床评价标准要到化疗进行到一定阶段,或肿瘤复发后,才能判断出患者对于化疗药物的敏感性,因此发现一种铂类耐药标记蛋白在患者接受化疗之前或治疗初期预知肿瘤的耐药性,及时调整化疗方案是解决这一问题的关键途径。
本课题组前期运用比较蛋白质组学技术筛选出了铂类耐药相关蛋白—膜联蛋白A3(Annexin A3, ANXA3),并在体外细胞实验、动物实验及临床研究中逐步验证了该指标和铂耐药的相关性、影响铂耐药的机制、血清可检测性以及在临床中作为铂耐药预测指标的可行性。因为市售Annexin A3ELISA试剂盒敏感性欠佳、重复性差,本课题组研制了具有较高灵敏性、可重复性的Annexin A3化学发光定量检测试剂盒,并在小样本的前瞻性临床研究中观察了该方法检测的治疗前Annexin A3血清浓度对铂类化疗耐药的诊断价值,发现该指标具有中等预测价值,ROC曲线下面积为0.744,最佳界值点为1.645,该界值的预测敏感度及特异度分别为66.70%、82.93%,阳性及阴性预测值分别为58.82%、87.18%。
本研究在现有研究的基础上前瞻性连续收集临床初治的卵巢癌患者的血清学和组织学标本,以化学发光免疫分析和免疫组化技术分别检测Annexin A3蛋白在观察对象外周血和肿瘤组织中的表达水平,评估Annexin A3蛋白在患者外周血和肿瘤组织中的表达水平对临床化疗耐药的预测作用及多指标联合检测提高预测价值的可行性,并对Annexin A3蛋白血清表达在复发上皮性卵巢癌患者中的临床意义进行了初步探讨。
研究方法
1、进一步扩大样本量,在较大样本患者中分析Annexin A3的血清浓度和铂类化疗耐药的关系；通过绘制ROC曲线评价其诊断价值,确定最佳界值点；并将扩大样本前后Annexin A3的血清浓度对铂类耐药的诊断价值进行比较。观察Annexin A3血清浓度对初治患者无进展生存的影响。
连续性收集初治上皮性卵巢癌患者治疗前血清标本及病例资料；采用AnnexinA3化学发光定量检测试剂盒测定收集标本的Annexin A3蛋白血清浓度并对其分布进行分析；通过查病例或电话回访随访患者的铂化疗结局；对Annexin A3蛋白血清浓度和临床病理特征进行Spearman相关性分析；观察铂类敏感及耐药组间AnnexinA3血清浓度的表达差别并通过Logistic多因素模型分析Annexin A3蛋白血清浓度和铂耐药的相关性；绘制ROC曲线评价Annexin A3蛋白血清浓度预测铂类耐药的价值,确定最佳界值点,观察该界值点的敏感度、特异度、阳性预测值及阴性预测值,并对扩大样本前后/Annexin A3蛋白血清浓度对铂类耐药的预测价值进行比较；在Cox多因素模型中分析Annexin A3血清水平对PFS的影响,并通过Log rank检验比较以最佳界值确定的Annexin A3高浓度组及低浓度组间PFS生存曲线的差别。
2、通过免疫组化技术检测Annexin A3蛋白在同一研究人群中的组织表达；分析Annexin A3蛋白的组织表达和铂类耐药的相关性；通过制作ROC曲线评价AnnexinA3蛋白对铂类耐药的预测价值；观察Annexin A3组织表达对PFS的影响；在同一研究人群中将Annexin A3蛋白的组织表达和血清浓度对铂类耐药的预测价值进行比较。
采用免疫组化方法对同一研究人群中肿瘤组织的Annexin A3蛋白表达进行半定量检测；将“阴性”及“+”表达患者定义为低表达组,将“++”及“+++”表达患者定义为高表达组,观察铂类敏感及耐药人群中Annexin A3蛋白的组织表达差异并通过多因素分析观察Annexin A3组织表达与铂化疗耐药的相关性；绘制ROC曲线计算曲线下面积评价Annexin A3组织表达对铂耐药的诊断价值,确定最佳界值点,观察该界值点的敏感性、特异性、阳性预测值及阴性预测值,并比较Annexin A3组织表达和血清浓度诊断铂耐药ROC曲线下面积的大小；通过多因素生存分析观察AnnexinA3组织表达对患者预后的影响并比较Annexin A3蛋白高表达组及低表达间PFS生存的差别。
3、通过体外细胞实验观察Cofilin-1蛋白作为铂耐药血清联合预测指标的可行性。稳定培养SKOV3、SKOV3/CDDP、COC1及COC1/CDDP细胞系；检测SKOV3/CDDP及COC1/CDDP两株耐药细胞系的耐药指数；通过Western Blot及免疫荧光实验比较Cofilin-1蛋白在耐药及敏感细胞株中的表达差异并对蛋白进行定位研究；浓缩细胞上清并通过Western Blot观察Cofilin-1是否在细胞上清中表达。
SKOV3及耐药株SKOV3/CDDP用DMEM (HG)培养基贴壁培养,COC1及其耐药株COC1/CDDP用RPMI1640培养基悬浮培养。运用MTT法检测SKOV3及SKOV3/CDDP细胞株的IC50,计算SKOV3/CDDP对SKOV3的耐药指数；运用CCK-8法检测COC1及COC1/CDDP细胞系的IC50,计算COC1/CDDP对COC1的耐药指数。待细胞处于对数生长期时,收集细胞裂解后BCA法测定蛋白浓度,运用Western Blot法对细胞的Cofilin-1蛋白表达水平进行半定量检测；通过免疫荧光法观察Cofilin-1蛋白在爬片细胞中的定位并比较敏感及耐药细胞中的荧光表达差别；用无血清培养基培养细胞,收集细胞培养上清后用超滤管离心浓缩蛋白,Western Blot法检测上清液中Cofilin-1蛋白的表达。
4、回顾性分析第二部分研究对象中未先期化疗患者术后首次化疗前后的CA125下降比率与铂类耐药的相关性；通过建立ROC曲线评估CA125下降比率及其和Annexin A3血清浓度联合对铂类耐药的预测价值；将联合检测及其单因子检测对铂类耐药的预测价值进行比较。
回顾性收集第二部分研究对象中未先期化疗患者术后首次化疗前后的CA125值,计算CA125下降比率并观察其分布情况；比较敏感及耐药人群中的CA125下降比率并对CA125下降比率和铂耐药之间的相关性进行Logistic多因素回归分析；制作CA125下降比率预测铂类耐药的ROC曲线,观察曲线下面积,并通过建立CA125下降比率和Annexin A3血清浓度预测铂类耐药的Logistic多因素回归模型,绘制两者联合检测的ROC曲线；将联合检测及单因子检测的曲线下面积进行比较。
5、用化学发光定量检测试剂盒检测复发上皮性卵巢癌患者治疗前血清的AnnexinA3蛋白水平；并对治疗前Annexin A3蛋白血清浓度和铂化疗结局的关系进行初步探讨。
连续收集复发上皮性卵巢癌患者治疗前的血清标本,随访患者治疗结局；采用化学发光定量检测试剂盒测定复发患者的Annexin A3蛋白血清浓度并观察其分布情况；将患者按无铂间期长短分成两组(<12月vs≥12月),观察每组患者治疗前Annexin A3蛋白的血清表达水平并进行组间比较；将有化疗结局的患者按铂化疗敏感及铂化疗耐药分组,观察Annexin A3蛋白血清水平的组间差异；观察化疗结束后不同时间点疾病进展和Annexin A3蛋白血清浓度的相关性；将患者按50百分位数分成Annexin A3蛋白高浓度组及低浓度组,比较两组间PFS生存的差别。
研究结果
1、自2009年9月至2012年4月期间在北京协和医院就诊的初治上皮性卵巢癌患者96例,采用化学发光试剂盒定量检测治疗前血清的Annexin A3蛋白水平,批内差异<15%,批间差异<20%。89例患者有铂类化疗结局,直方图及P-P图显示其Annexin A3血清浓度呈非正态分布,Kolmogorov-SmirnovZ检验Z值为1.475,P值为0.026；89例患者的浓度范围0.20ng/mL～6.18ng/mL,中位浓度为1.20ng/mL,四分位间距为1.62ng/mL。截止2013年3月,3例(3.13%)失访,患者中位随访时间为14月(7月-39月)；铂类化疗敏感患者68例,铂类化疗耐药(包括难治)患者21例。68例铂敏感患者治疗前外周血Annexin A3蛋白浓度为1.33±0.91ng/mL,显著高于铂耐药患者的2.28±1.69ng/mL (P=0.001); Logistic多因素分析显示Annexin A3血清浓度及手术彻底性与铂类化疗耐药/难治有显著相关性,P值分别为0.028及0.016。治疗前Annexin A3血清水平预测铂类耐药的ROC曲线曲线下面积为0.733,最佳界值为2.05ng/mL时,该界值的敏感度为61.11%,特异度为80.88%,阳性预测值为45.8%,阴性预测值为88.7%；扩大样本后的ROC曲线下面积较预实验略增加。Annexin A3血清高表达组(≥2.05ng/mL)的中位PFS时间较低浓度组(<2.05ng/mL)长(14月vs11.5月),COX多因素分析显示Annexin A3血清浓度(P=0.002)和手术彻底性(P=0.001)对PFS有显著影响,log-rank检验显示AnnexinA3血清高浓度组及低浓度组的PFS生存差异显著(P<0.05)。
2、89例初治上皮性卵巢癌患者中38(42.7%)例Annexin A3阴性表达,30(33.7%)例表达为“+”,15(16.9%)例表达为“++”,6(6.7%)例表达为“+++”；68例(75.3%)患者低表达,21(24.7%)例患者高表达。Spearman相关性分析显示Annexin A3组织表达和和铂耐药相关性显著(P=0.012),相关系数为0.266。铂耐药患者中高表达患者占42.9%,显著高于铂敏感人群的17.6%(P=0.017)。多因素分析显示/Annexin A3组织表达(低表达&高表达,P=0.004)及手术彻底性(P=0.002)和铂耐药显著相关。Annexin A3组织表达预测铂类耐药的ROC曲线下面积为0.664,当Annexin A3组织表达≥++时,约登指数最大,此界值的敏感度及特异度分别为42.86%及82.35%,阳性预测值及阴性预测值分别为58.82%及87.18%。Annexin A3组织表达预测铂耐药的ROC曲线下面积小于Annexin A3血清浓度的ROC曲线下面积,但差异无显著性(0.664&0.773,P=0.544)。多因素生存分析显示Annexin A3组织表达及手术彻底性对PFS影响显著(P值分别为0.010及0.004),Annexin A3(?)表达组的PFS时间较高表达组长(14月vs11月),Log-rank检验显示Annexin A3高表达及低表达组间PFS生存有显著差异(P<0.001)。
3、SKOV3、SKOV3/CDDP、COC1及COC1/CDDP细胞生长状态良好,SKOV3/CDDP对SKOV3的耐药指数平均为6.40±1.55,COC1/CDDP对COC1的耐药指数平均为2.87±1.16。Western blot半定量检测显示Cofilin1蛋白在SKOV3/CDDP耐药细胞胞浆中的表达高于SKOV3敏感细胞株,在COC1/CDDP胞浆中的表达高于COC1细胞株,其中以SKOV3耐药及敏感株中的Cofilin1蛋白差异更为明显。细胞免疫荧光观察发现Cofilinl蛋白定位于细胞胞浆,两种细胞系中耐药株的荧光强度均较敏感株强。细胞培养上清浓缩后western blot未检测到Cofilinl蛋白的表达。
4、第二部分研究对象中首次化疗前后CA125资料完整的未先期化疗卵巢癌患者61例,其中铂敏感患者48例,铂耐药患者13例。61例患者CA125下降率其总体分布呈非正态分布,Kolmogorov-SmirnovZ检验Z值为2.892,P<0.001。铂耐药患者的CA125下降比率中位数0.25显著低于铂敏感组的0.64(P=0.001)。去除1例极值,多因素分析显示CA125下降比率及治疗前Annexin A3血清浓度为铂耐药的显著相关因素(P值分别为0.026及0.028)。CA125下降比率诊断铂耐药的ROC曲线下面积为0.813,最佳界值点为0.577,此点的敏感度及特异度为92.31%及61.70%,阳性及阴性预测值分别为40.0%,为96.7%。设铂耐药发生风险值Y,建立AnnexinA3血清浓度及CA125下降率预测铂耐药的Logistic回归模型LogY=-1.83+0.938×Annexin A3血清浓度-2.674×CA125变化率,Y值诊断铂耐药的ROC曲线下面积为0.863,最佳界值点的敏感度及特异度分别为92.31%及74.47%,阳性及阴性预测值分别为50.0%及97.2%。该联合检测的ROC曲线下面积大于Annexin A3血清浓度单因子预测的曲线下面积(0.863&0.745),P值为0.053。
5、纳入符合标准的的复发性上皮性卵巢癌患者61例,其中54例无铂间期≥6月,7例无铂间期<6月。铂化疗难治性患者9例,铂类化疗耐药患者8例,铂类化疗敏感患者7例。化学发光法检测治疗前Annexin A3蛋白血清浓度板内变异系数小于15%,板间变异系数小于20%。直方图及P-P图提示Annexin A3蛋白血清浓度呈正态分布(Z=265,P=.082),平均浓度为0.77±0.56ng/mL。Spearman相关性分析显示治疗前Annexin A3血清浓度和铂类化疗耐药无显著相关性,P值为0.517。无铂间期<12月及≥12月的患者平均浓度为0.92±0.60ng/ml及0.67±0.50ng/ml,差异无显著性(P=0.08)。17例例铂耐药患者的中位Annexin A3血清浓度高于7例铂敏感患者(0.87ng/mL vs0.67ng/mL),但组间差异无显著性(P=0.314)。化疗结束各时间点疾病进展患者的血清浓度高于疾病无进展患者血清浓度,差别无显著性。Annexin A3蛋白高浓度组的中位PFS时间较低浓度组短(6月vs7月),PFS生存比较无显著差异(P=0.09)。
结论
1、初治上皮性卵巢癌患者的外周血Annexin A3蛋白表达水平和铂耐药相关性显著,对临床铂类耐药有中等预测价值,最佳界值为2.05ng/mL。Annexin A3血清浓度及手术彻底性对铂类耐药及无进展生存均有显著的影响。
2、初治上皮性卵巢癌患者肿瘤组织的Annexin A3蛋白表达和铂耐药显著相关,对铂耐药预测价值低；Annexin A3蛋白组织表达预测铂类耐药的ROC曲线下面积小于Annexin A3血清浓度,但差异无显著性；Annexin A3蛋白组织表达是无进展生存的显著影响因素。
3、体外细胞实验证实Cofilin-1蛋白和铂类耐药相关,但是不具有细胞外泌性,因此不能作为铂类耐药预测的血清联合检测指标。
4、CA125下降比率和铂耐药有显著相关性,外周血Annexin A3蛋白表达和CA125下降比率联合对铂耐药的预测价值优于单因子预测,CA125下降比率作为铂耐药的血清联合预测指标具有可行性。
5、小样本初步研究显示复发上皮卵巢癌患者的治疗前Annexin A3蛋白血清水平和铂类耐药相关性不显著,但是铂耐药患者的治疗前Annexin A3蛋白血清浓度略高于铂敏感患者。
Background and Purpose
Epithelia 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.
Methods
1、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.
Results
1、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)
Conclusions
1、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.
卵巢上皮癌是女性生殖器官中死亡率居第一位的恶性肿瘤,目前的标准治疗方案是在肿瘤细胞减灭术的基础上辅以紫杉醇和铂类联合的一线化疗,但是预后不理想,晚期患者的5年生存率仍然徘徊在30%左右。肿瘤细胞对于化疗药物产生耐药性是造成卵巢癌的5年生存率得不到进一步改善的主要原因之一,初治患者的完全缓解率接近75%,仍有15-30%的患者对初治方案反应不佳；在达到完全缓解的患者中,有>90%的患者肿瘤复发。如何早期发现和诊断化疗耐药,进一步改善卵巢癌的预后是当前困扰妇科肿瘤临床的主要问题之一。卵巢癌患者对铂类药物耐药的临床评价标准要到化疗进行到一定阶段,或肿瘤复发后,才能判断出患者对于化疗药物的敏感性,因此发现一种铂类耐药标记蛋白在患者接受化疗之前或治疗初期预知肿瘤的耐药性,及时调整化疗方案是解决这一问题的关键途径。
本课题组前期运用比较蛋白质组学技术筛选出了铂类耐药相关蛋白—膜联蛋白A3(Annexin A3, ANXA3),并在体外细胞实验、动物实验及临床研究中逐步验证了该指标和铂耐药的相关性、影响铂耐药的机制、血清可检测性以及在临床中作为铂耐药预测指标的可行性。因为市售Annexin A3ELISA试剂盒敏感性欠佳、重复性差,本课题组研制了具有较高灵敏性、可重复性的Annexin A3化学发光定量检测试剂盒,并在小样本的前瞻性临床研究中观察了该方法检测的治疗前Annexin A3血清浓度对铂类化疗耐药的诊断价值,发现该指标具有中等预测价值,ROC曲线下面积为0.744,最佳界值点为1.645,该界值的预测敏感度及特异度分别为66.70%、82.93%,阳性及阴性预测值分别为58.82%、87.18%。
本研究在现有研究的基础上前瞻性连续收集临床初治的卵巢癌患者的血清学和组织学标本,以化学发光免疫分析和免疫组化技术分别检测Annexin A3蛋白在观察对象外周血和肿瘤组织中的表达水平,评估Annexin A3蛋白在患者外周血和肿瘤组织中的表达水平对临床化疗耐药的预测作用及多指标联合检测提高预测价值的可行性,并对Annexin A3蛋白血清表达在复发上皮性卵巢癌患者中的临床意义进行了初步探讨。
研究方法
1、进一步扩大样本量,在较大样本患者中分析Annexin A3的血清浓度和铂类化疗耐药的关系；通过绘制ROC曲线评价其诊断价值,确定最佳界值点；并将扩大样本前后Annexin A3的血清浓度对铂类耐药的诊断价值进行比较。观察Annexin A3血清浓度对初治患者无进展生存的影响。
连续性收集初治上皮性卵巢癌患者治疗前血清标本及病例资料；采用AnnexinA3化学发光定量检测试剂盒测定收集标本的Annexin A3蛋白血清浓度并对其分布进行分析；通过查病例或电话回访随访患者的铂化疗结局；对Annexin A3蛋白血清浓度和临床病理特征进行Spearman相关性分析；观察铂类敏感及耐药组间AnnexinA3血清浓度的表达差别并通过Logistic多因素模型分析Annexin A3蛋白血清浓度和铂耐药的相关性；绘制ROC曲线评价Annexin A3蛋白血清浓度预测铂类耐药的价值,确定最佳界值点,观察该界值点的敏感度、特异度、阳性预测值及阴性预测值,并对扩大样本前后/Annexin A3蛋白血清浓度对铂类耐药的预测价值进行比较；在Cox多因素模型中分析Annexin A3血清水平对PFS的影响,并通过Log rank检验比较以最佳界值确定的Annexin A3高浓度组及低浓度组间PFS生存曲线的差别。
2、通过免疫组化技术检测Annexin A3蛋白在同一研究人群中的组织表达；分析Annexin A3蛋白的组织表达和铂类耐药的相关性；通过制作ROC曲线评价AnnexinA3蛋白对铂类耐药的预测价值；观察Annexin A3组织表达对PFS的影响；在同一研究人群中将Annexin A3蛋白的组织表达和血清浓度对铂类耐药的预测价值进行比较。
采用免疫组化方法对同一研究人群中肿瘤组织的Annexin A3蛋白表达进行半定量检测；将“阴性”及“+”表达患者定义为低表达组,将“++”及“+++”表达患者定义为高表达组,观察铂类敏感及耐药人群中Annexin A3蛋白的组织表达差异并通过多因素分析观察Annexin A3组织表达与铂化疗耐药的相关性；绘制ROC曲线计算曲线下面积评价Annexin A3组织表达对铂耐药的诊断价值,确定最佳界值点,观察该界值点的敏感性、特异性、阳性预测值及阴性预测值,并比较Annexin A3组织表达和血清浓度诊断铂耐药ROC曲线下面积的大小；通过多因素生存分析观察AnnexinA3组织表达对患者预后的影响并比较Annexin A3蛋白高表达组及低表达间PFS生存的差别。
3、通过体外细胞实验观察Cofilin-1蛋白作为铂耐药血清联合预测指标的可行性。稳定培养SKOV3、SKOV3/CDDP、COC1及COC1/CDDP细胞系；检测SKOV3/CDDP及COC1/CDDP两株耐药细胞系的耐药指数；通过Western Blot及免疫荧光实验比较Cofilin-1蛋白在耐药及敏感细胞株中的表达差异并对蛋白进行定位研究；浓缩细胞上清并通过Western Blot观察Cofilin-1是否在细胞上清中表达。
SKOV3及耐药株SKOV3/CDDP用DMEM (HG)培养基贴壁培养,COC1及其耐药株COC1/CDDP用RPMI1640培养基悬浮培养。运用MTT法检测SKOV3及SKOV3/CDDP细胞株的IC50,计算SKOV3/CDDP对SKOV3的耐药指数；运用CCK-8法检测COC1及COC1/CDDP细胞系的IC50,计算COC1/CDDP对COC1的耐药指数。待细胞处于对数生长期时,收集细胞裂解后BCA法测定蛋白浓度,运用Western Blot法对细胞的Cofilin-1蛋白表达水平进行半定量检测；通过免疫荧光法观察Cofilin-1蛋白在爬片细胞中的定位并比较敏感及耐药细胞中的荧光表达差别；用无血清培养基培养细胞,收集细胞培养上清后用超滤管离心浓缩蛋白,Western Blot法检测上清液中Cofilin-1蛋白的表达。
4、回顾性分析第二部分研究对象中未先期化疗患者术后首次化疗前后的CA125下降比率与铂类耐药的相关性；通过建立ROC曲线评估CA125下降比率及其和Annexin A3血清浓度联合对铂类耐药的预测价值；将联合检测及其单因子检测对铂类耐药的预测价值进行比较。
回顾性收集第二部分研究对象中未先期化疗患者术后首次化疗前后的CA125值,计算CA125下降比率并观察其分布情况；比较敏感及耐药人群中的CA125下降比率并对CA125下降比率和铂耐药之间的相关性进行Logistic多因素回归分析；制作CA125下降比率预测铂类耐药的ROC曲线,观察曲线下面积,并通过建立CA125下降比率和Annexin A3血清浓度预测铂类耐药的Logistic多因素回归模型,绘制两者联合检测的ROC曲线；将联合检测及单因子检测的曲线下面积进行比较。
5、用化学发光定量检测试剂盒检测复发上皮性卵巢癌患者治疗前血清的AnnexinA3蛋白水平；并对治疗前Annexin A3蛋白血清浓度和铂化疗结局的关系进行初步探讨。
连续收集复发上皮性卵巢癌患者治疗前的血清标本,随访患者治疗结局；采用化学发光定量检测试剂盒测定复发患者的Annexin A3蛋白血清浓度并观察其分布情况；将患者按无铂间期长短分成两组(<12月vs≥12月),观察每组患者治疗前Annexin A3蛋白的血清表达水平并进行组间比较；将有化疗结局的患者按铂化疗敏感及铂化疗耐药分组,观察Annexin A3蛋白血清水平的组间差异；观察化疗结束后不同时间点疾病进展和Annexin A3蛋白血清浓度的相关性；将患者按50百分位数分成Annexin A3蛋白高浓度组及低浓度组,比较两组间PFS生存的差别。
研究结果
1、自2009年9月至2012年4月期间在北京协和医院就诊的初治上皮性卵巢癌患者96例,采用化学发光试剂盒定量检测治疗前血清的Annexin A3蛋白水平,批内差异<15%,批间差异<20%。89例患者有铂类化疗结局,直方图及P-P图显示其Annexin A3血清浓度呈非正态分布,Kolmogorov-SmirnovZ检验Z值为1.475,P值为0.026；89例患者的浓度范围0.20ng/mL～6.18ng/mL,中位浓度为1.20ng/mL,四分位间距为1.62ng/mL。截止2013年3月,3例(3.13%)失访,患者中位随访时间为14月(7月-39月)；铂类化疗敏感患者68例,铂类化疗耐药(包括难治)患者21例。68例铂敏感患者治疗前外周血Annexin A3蛋白浓度为1.33±0.91ng/mL,显著高于铂耐药患者的2.28±1.69ng/mL (P=0.001); Logistic多因素分析显示Annexin A3血清浓度及手术彻底性与铂类化疗耐药/难治有显著相关性,P值分别为0.028及0.016。治疗前Annexin A3血清水平预测铂类耐药的ROC曲线曲线下面积为0.733,最佳界值为2.05ng/mL时,该界值的敏感度为61.11%,特异度为80.88%,阳性预测值为45.8%,阴性预测值为88.7%；扩大样本后的ROC曲线下面积较预实验略增加。Annexin A3血清高表达组(≥2.05ng/mL)的中位PFS时间较低浓度组(<2.05ng/mL)长(14月vs11.5月),COX多因素分析显示Annexin A3血清浓度(P=0.002)和手术彻底性(P=0.001)对PFS有显著影响,log-rank检验显示AnnexinA3血清高浓度组及低浓度组的PFS生存差异显著(P<0.05)。
2、89例初治上皮性卵巢癌患者中38(42.7%)例Annexin A3阴性表达,30(33.7%)例表达为“+”,15(16.9%)例表达为“++”,6(6.7%)例表达为“+++”；68例(75.3%)患者低表达,21(24.7%)例患者高表达。Spearman相关性分析显示Annexin A3组织表达和和铂耐药相关性显著(P=0.012),相关系数为0.266。铂耐药患者中高表达患者占42.9%,显著高于铂敏感人群的17.6%(P=0.017)。多因素分析显示/Annexin A3组织表达(低表达&高表达,P=0.004)及手术彻底性(P=0.002)和铂耐药显著相关。Annexin A3组织表达预测铂类耐药的ROC曲线下面积为0.664,当Annexin A3组织表达≥++时,约登指数最大,此界值的敏感度及特异度分别为42.86%及82.35%,阳性预测值及阴性预测值分别为58.82%及87.18%。Annexin A3组织表达预测铂耐药的ROC曲线下面积小于Annexin A3血清浓度的ROC曲线下面积,但差异无显著性(0.664&0.773,P=0.544)。多因素生存分析显示Annexin A3组织表达及手术彻底性对PFS影响显著(P值分别为0.010及0.004),Annexin A3(?)表达组的PFS时间较高表达组长(14月vs11月),Log-rank检验显示Annexin A3高表达及低表达组间PFS生存有显著差异(P<0.001)。
3、SKOV3、SKOV3/CDDP、COC1及COC1/CDDP细胞生长状态良好,SKOV3/CDDP对SKOV3的耐药指数平均为6.40±1.55,COC1/CDDP对COC1的耐药指数平均为2.87±1.16。Western blot半定量检测显示Cofilin1蛋白在SKOV3/CDDP耐药细胞胞浆中的表达高于SKOV3敏感细胞株,在COC1/CDDP胞浆中的表达高于COC1细胞株,其中以SKOV3耐药及敏感株中的Cofilin1蛋白差异更为明显。细胞免疫荧光观察发现Cofilinl蛋白定位于细胞胞浆,两种细胞系中耐药株的荧光强度均较敏感株强。细胞培养上清浓缩后western blot未检测到Cofilinl蛋白的表达。
4、第二部分研究对象中首次化疗前后CA125资料完整的未先期化疗卵巢癌患者61例,其中铂敏感患者48例,铂耐药患者13例。61例患者CA125下降率其总体分布呈非正态分布,Kolmogorov-SmirnovZ检验Z值为2.892,P<0.001。铂耐药患者的CA125下降比率中位数0.25显著低于铂敏感组的0.64(P=0.001)。去除1例极值,多因素分析显示CA125下降比率及治疗前Annexin A3血清浓度为铂耐药的显著相关因素(P值分别为0.026及0.028)。CA125下降比率诊断铂耐药的ROC曲线下面积为0.813,最佳界值点为0.577,此点的敏感度及特异度为92.31%及61.70%,阳性及阴性预测值分别为40.0%,为96.7%。设铂耐药发生风险值Y,建立AnnexinA3血清浓度及CA125下降率预测铂耐药的Logistic回归模型LogY=-1.83+0.938×Annexin A3血清浓度-2.674×CA125变化率,Y值诊断铂耐药的ROC曲线下面积为0.863,最佳界值点的敏感度及特异度分别为92.31%及74.47%,阳性及阴性预测值分别为50.0%及97.2%。该联合检测的ROC曲线下面积大于Annexin A3血清浓度单因子预测的曲线下面积(0.863&0.745),P值为0.053。
5、纳入符合标准的的复发性上皮性卵巢癌患者61例,其中54例无铂间期≥6月,7例无铂间期<6月。铂化疗难治性患者9例,铂类化疗耐药患者8例,铂类化疗敏感患者7例。化学发光法检测治疗前Annexin A3蛋白血清浓度板内变异系数小于15%,板间变异系数小于20%。直方图及P-P图提示Annexin A3蛋白血清浓度呈正态分布(Z=265,P=.082),平均浓度为0.77±0.56ng/mL。Spearman相关性分析显示治疗前Annexin A3血清浓度和铂类化疗耐药无显著相关性,P值为0.517。无铂间期<12月及≥12月的患者平均浓度为0.92±0.60ng/ml及0.67±0.50ng/ml,差异无显著性(P=0.08)。17例例铂耐药患者的中位Annexin A3血清浓度高于7例铂敏感患者(0.87ng/mL vs0.67ng/mL),但组间差异无显著性(P=0.314)。化疗结束各时间点疾病进展患者的血清浓度高于疾病无进展患者血清浓度,差别无显著性。Annexin A3蛋白高浓度组的中位PFS时间较低浓度组短(6月vs7月),PFS生存比较无显著差异(P=0.09)。
结论
1、初治上皮性卵巢癌患者的外周血Annexin A3蛋白表达水平和铂耐药相关性显著,对临床铂类耐药有中等预测价值,最佳界值为2.05ng/mL。Annexin A3血清浓度及手术彻底性对铂类耐药及无进展生存均有显著的影响。
2、初治上皮性卵巢癌患者肿瘤组织的Annexin A3蛋白表达和铂耐药显著相关,对铂耐药预测价值低；Annexin A3蛋白组织表达预测铂类耐药的ROC曲线下面积小于Annexin A3血清浓度,但差异无显著性；Annexin A3蛋白组织表达是无进展生存的显著影响因素。
3、体外细胞实验证实Cofilin-1蛋白和铂类耐药相关,但是不具有细胞外泌性,因此不能作为铂类耐药预测的血清联合检测指标。
4、CA125下降比率和铂耐药有显著相关性,外周血Annexin A3蛋白表达和CA125下降比率联合对铂耐药的预测价值优于单因子预测,CA125下降比率作为铂耐药的血清联合预测指标具有可行性。
5、小样本初步研究显示复发上皮卵巢癌患者的治疗前Annexin A3蛋白血清水平和铂类耐药相关性不显著,但是铂耐药患者的治疗前Annexin A3蛋白血清浓度略高于铂敏感患者。
Background and Purpose
Epithelia 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.
Methods
1、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.
Results
1、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)
Conclusions
1、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.
引文
[1]Jemal A S, R.Xu, J.Ward, E. Cancer statistics,2010 [J]. CA:a cancer journal for clinicians,2010,60(5):277-300.
[2]Hernandez B Y G, M. D.Cassel, K. D.Pobutsky, A. M., Vu V, Wilkens L R. Preview of Hawaii Cancer Facts and Figures 2010 [J]. Hawaii medical journal,2010,69(9): 223-4.
[3]Bookman M A B, M. F.McGuire, W. P.Harper, P. G.Alberts, D. S.Friedlander, M., Colombo N, Fowler J M, et al. Evaluation of new platinum-based treatment regimens in advanced-stage ovarian cancer:a Phase Ⅲ Trial of the Gynecologic Cancer Intergroup [J]. Journal of clinical oncology:official journal of the American Society of Clinical Oncology,2009,27(9):1419-25.
[4]Yan X D P, L. Y.Yuan, Y.Lang, J. H., Mao N. Identification of platinum-resistance associated proteins through proteomic analysis of human ovarian cancer cells and their platinum-resistant sublines [J]. Journal of proteome research,2007,6(2):772-80.
[5]Moss S E M, R. O. The annexins [J]. Genome biology,2004,5(4):219.
[6]Mussunoor S M, G. I. The role of annexins in tumour development and progression [J]. The Journal of pathology,2008,216(2):131-40.
[7]Tait J F F, D. A.Miao, C. H.Killary, A. M.Adler, D. A., Disteche C M. Chromosomal localization of the human annexin Ⅲ (ANX3) gene [J]. Genomics,1991,10(2):441-8.
[8]Cao D L Y, D. W.Zhang, H. L.Zhu, Y, Wang Y X, Yao X D. 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]. The Prostate,2011,71(7): 700-10.
[9]Bianchi C B, S.Raimondo, F.Torsello, B.Angeloni, V., Ferrero S, Di Stefano V, 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]. The American journal of pathology,2010,176(4):1660-70.
[10]Yin J Y, 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]. Journal of cellular and molecular medicine,2012,16(2):337-48.
[11]Kollermann J S, T.Bang, H.Schwall, G. P.von Eichel-Streiber, C, Simon R, Schostak M, et al. Expression and prognostic relevance of annexin A3 in prostate cancer [J]. European urology,2008,54(6):1314-23.
[12]赵洁魏.免疫组化技术的标准化质控管理[J].青岛大学医学院学报,2009,45(2)：175-6.
[13]Han Y H, H.Xiao, Z.Zhang, W.Cao, Y.Qu, L.Shou, C. Integrated analysis of gene expression profiles associated with response of platinum/paclitaxel-based treatment in epithelial ovarian cancer [J]. PLoS One,2012,7(12):e52745.
[14]E1-Rifai W, J. StuartKim, YoungchulDuska, LindaBirrer, Michael, Levine D A, Moskaluk C, et al. Multi-Gene Expression Predictors of Single Drug Responses to Adjuvant Chemotherapy in Ovarian Carcinoma:Predicting Platinum Resistance [J]. PLoS ONE,2012,7(2):e30550.
[15]DesMarais V, Ghosh M, Eddy R, et al. Cofilin takes the lead [J]. Journal of cell science,2005,118(Pt 1):19-26.
[16]Nishimura S, Tsuda H, Kataoka F, et al. Overexpression of cofilin 1 can predict progression-free survival in patients with epithelial ovarian cancer receiving standard therapy [J]. Human pathology,2011,42(4):516-21.
[17]Riedinger J M, Bonnetain F, Basuyau J P, et al. Change in CA125 levels after the first cycle of induction chemotherapy is an independent predictor of epithelial ovarian tumour outcome [J]. Annals of oncology:official journal of the European Society for Medical Oncology/ESMO,2007,18(5):881-5.
[18]Karam A K K, B. Y. Ovarian cancer:the duplicity of CA125 measurement [J]. Nature reviews Clinical oncology,2010,7(6):335-9.
[19]Therasse P A, S. G.Eisenhauer, E. A.Wanders, J.Kaplan, R. S.Rubinstein, L., Verweij J, Van Glabbeke M, et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada [J]. Journal of the National Cancer Institute,2000,92(3):205-16.
[20]Galaal K N R, Bristow RE, Patel A, Bryant A, DickinsonHO. Cytoreductive surgery plus chemotherapy versus chemotherapy alone for recurrent epithelial ovarian cancer [J]. TheCochrane Library,2010,6):
[21]Colombo N, Gore M. Treatment of recurrent ovarian cancer relapsing 6-12 months post platinum-based chemotherapy [J]. Critical reviews in oncology/hematology,2007, 64(2):129-38.
[22]Zajchowski D A, Karlan B Y, Shawver L K. Treatment-Related Protein Biomarker Expression Differs between Primary and Recurrent Ovarian Carcinomas [J]. Molecular Cancer Therapeutics,2011,11 (2):492-502.
[23]Outcome of ATP-based tumor chemosensitivity assay directed chemotherapy in heavily pre-treated recurrent ovarian carcinoma [J].
[24]Melichar B F, P.Tomsova, M.Malirova, E. Repeat chemosensitivity of epithelial ovarian carcinoma in a BRCA1 mutation carrier to paclitaxel/platinum combination chemotherapy [J]. European journal of gynaecological oncology,2009,30(3):323-5.
[1]Hernandez B Y G, M. D.Cassel, K. D.Pobutsky, A. M., Vu V, Wilkens L R. Preview of Hawaii Cancer Facts and Figures 2010 [J]. Hawaii medical journal,2010,69(9): 223-4.
[2]Tagawa T, Robert Yen, Yun, Mortimer J. Ovarian Cancer:Opportunity for Targeted Therapy [J]. Journal of Oncology,2012,2012(1-9.
[3]McGuire W P H, W. J.Brady, M. F.Kucera, P. R.Partridge, E. E.Look, K. Y, Clarke-Pearson D L, Davidson M. Cyclophosphamide and cisplatin compared with paclitaxel and cisplatin in patients with stage Ⅲ and stage Ⅳ ovarian cancer [J]. The New England journal of medicine,1996,334(1):1-6.
[4]Ledermann J A K, R. S. Optimal treatment for relapsing ovarian cancer [J]. Annals of oncology:official journal of the European Society for Medical Oncology/ESMO, 2010,21 Supp17(vii218-22.
[5]Kartalou M E, J. M. Mechanisms of resistance to cisplatin [J]. Mutation research, 2001,478(1-2):23-43.
[6]Jandial D D F-H, S.Larson, C. A., Elliott G I, Wrasidlo W J, et al. Enhanced delivery of cisplatin to intraperitoneal ovarian carcinomas mediated by the effects of bortezomib on the human copper transporter 1 [J]. Clinical cancer research:an official journal of the American Association for Cancer Research,2009,15(2):553-60.
[7]Liu J J, Jamieson S M, Subramaniam J, et al. Neuronal expression of copper transporter 1 in rat dorsal root ganglia:association with platinum neurotoxicity [J]. Cancer Chemother Pharmacol,2009,64(4):847-56.
[8]Borst P E, R.Kool, M.Wijnholds, J. A family of drug transporters:the multidrug resistance-associated proteins [J]. Journal of the National Cancer Institute,2000,92(16): 1295-302.
[9]Liedert B M, V.Schadendorf, D.Thomale, J., Lage H. Overexpression of cMOAT (MRP2/ABCC2) is associated with decreased formation of platinum-DNA adducts and decreased G2-arrest in melanoma cells resistant to cisplatin [J]. The Journal of investigative dermatology,2003,121(1):172-6.
[10]Mangala L S Z, V.Schmandt, R.Leshane, E. S.Halder, J. B., Armaiz-Pena G N, Spannuth W A, et al. Therapeutic Targeting of ATP7B in Ovarian Carcinoma [J]. Clinical cancer research:an official journal of the American Association for Cancer Research, 2009,15(11):3770-80.
[11]Halder J K, A. A.Landen, C. N., Jr.Han, L. Y.Lutgendorf, S. K., Lin Y G, Merritt W M, et al. Focal adhesion kinase targeting using in vivo short interfering RNA delivery in neutral liposomes for ovarian carcinoma therapy [J]. Clinical cancer research:an official journal of the American Association for Cancer Research,2006,12(16):4916-24.
[12]Nakayama K K, A.Ogawa, K.Miyazaki, K.Neamati, N., Takebayashi Y. Copper-transporting P-type adenosine triphosphatase (ATP7B) as a cisplatin based chemoresistance marker in ovarian carcinoma:comparative analysis with expression of MDR1, MRP1, MRP2, LRP and BCRP [J]. International journal of cancer Journal international du cancer,2002,101(5):488-95.
[13]Lewis A D H, J. D.Wolf, C. R. Glutathione and glutathione-dependent enzymes in ovarian adenocarcinoma cell lines derived from a patient before and after the onset of drug resistance:intrinsic differences and cell cycle effects [J]. Carcinogenesis,1988,9(7): 1283-7.
[14]Kang S J, W.Kim, J. W.Park, N. H.Song, Y. S.Kim, S. C., Park S Y, Kang S B, et al. Association between excision repair cross-complementation group 1 polymorphism and clinical outcome of platinum-based chemotherapy in patients with epithelial ovarian cancer [J]. Experimental & molecular medicine,2006,38(3):320-4.
[15]Steffensen K D W, M.Jakobsen, A. The relationship of platinum resistance and ERCC1 protein expression in epithelial ovarian cancer [J]. International journal of gynecological cancer:official journal of the International Gynecological Cancer Society, 2009,19(5):820-5.
[16]Rubatt J M, Darcy K M, Tian C, et al. Pre-treatment tumor expression of ERCC1 in women with advanced stage epithelial ovarian cancer is not predictive of clinical outcomes:a Gynecologic Oncology Group study [J]. Gynecol Oncol,2012,125(2): 421-6.
[17]Brown R H, G. L.Gallagher, W. M.McIlwrath, A. J.Margison, G. P.van der Zee, A. G., Anthoney D A. hMLHl expression and cellular responses of ovarian tumour cells to treatment with cytotoxic anticancer agents [J]. Oncogene,1997,15(1):45-52.
[18]Kamal N S S, J. C.Mendiboure, J.Planchard, D.Olaussen, K. A.Rousseau, V., Popper H, Pirker R, et al. MutS homologue 2 and the long-term benefit of adjuvant chemotherapy in lung cancer [J]. Clinical cancer research:an official journal of the American Association for Cancer Research,2010,16(4):1206-15.
[19]Shachar S, Ziv O, Avkin S, et al. Two-polymerase mechanisms dictate error-free and error-prone translesion DNA synthesis in mammals [J]. The EMBO journal,2009,28(4): 383-93.
[20]Galluzzi L S, L.Vitale, I.Michels, J., Martins I, Kepp O, et al. Molecular mechanisms of cisplatin resistance [J]. Oncogene,2012,31(15):1869-83.
[21]Narod S A F, W. D. BRCA1 and BRCA2:1994 and beyond [J]. Nature reviews Cancer,2004,4(9):665-76.
[22]Gourley C M, C. O.Roxburgh, P.Yap, T. A.Harden, S., Paul J, Ragupathy K, et al. Increased incidence of visceral metastases in Scottish patients with BRCA1/2-defective ovarian cancer:an extension of the ovarian BRCAness phenotype [J]. Journal of clinical oncology:official journal of the American Society of Clinical Oncology,2010,28(15): 2505-11.
[23]Dann R B D, J. A.Timms, K. M.Zom, K. K., Potter J, Flake D D,2nd, et al. BRCA1/2 mutations and expression:response to platinum chemotherapy in patients with advanced stage epithelial ovarian cancer [J]. Gynecol Oncol,2012,125(3):677-82.
[24]Gadducci A C, S.Muraca, S.Genazzani, A. R. Molecular mechanisms of apoptosis and chemosensitivity to platinum and paclitaxel in ovarian cancer:biological data and clinical implications [J]. European journal of gynaecological oncology,2002,23(5): 390-6.
[25]Williams J L, P. C.Griffith, K. A.Choi, M.Fogoros, S., Hu Y Y, Liu J R. Expression of Bcl-xL in ovarian carcinoma is associated with chemoresistance and recurrent disease [J]. Gynecol Oncol,2005,96(2):287-95.
[26]Chen L L, L.Yan, X.Liu, N.Gong, L., Pan S, Lin F, et al. Survivin status affects prognosis and chemosensitivity in epithelial ovarian cancer [J]. International journal of gynecological cancer:official journal of the International Gynecological Cancer Society, 2013,23(2):256-63.
[27]Hu J F, E. Depleting Mirk Kinase Increases Cisplatin Toxicity in Ovarian Cancer Cells [J]. Genes & cancer,2010,1(8):803-11.
[28]Yu H S, J.Xu, Y.Kang, J., Li H, Zhang L, et al. p62/SQSTM1 involved in cisplatin resistance in human ovarian cancer cells by clearing ubiquitinated proteins [J]. European journal of cancer (Oxford, England:1990),2011,47(10):1585-94.
[29]Sherman-Baust C A W, A. T.Rangel, L. B., Pizer E S, Cho K R, et al. Remodeling of the extracellular matrix through overexpression of collagen VI contributes to cisplatin resistance in ovarian cancer cells [J]. Cancer cell,2003,3(4):377-86.
[30]Pan S C, L.White, J. T.Lu, W.Utleg, A. G., Yan X, Urban N D, et al. Quantitative proteomics analysis integrated with microarray data reveals that extracellular matrix proteins, catenins, and p53 binding protein 1 are important for chemotherapy response in ovarian cancers [J]. OMICS,2009,13(4):345-54.
[31]Bartel D P. MicroRNAs:genomics, biogenesis, mechanism, and function [J]. Cell, 2004,116(2):281-97.
[32]Ma L T-F, J.Weinberg, R. A. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer [J]. Nature,2007,449(7163):682-8.
[33]Climent J D, P.Fridlyand, J.Palacios, J.Siebert, R.Albertson, D. G., Gray J W, Pinkel D, et al. Deletion of chromosome 11q predicts response to anthracycline-based chemotherapy in early breast cancer [J]. Cancer Res,2007,67(2):818-26.
[34]Blower P E C, J. H.Verducci, J. S.Lin, S.Park, J. K.Dai, Z.Liu, C. G., Schmittgen T D, Reinhold W C, et al. MicroRNAs modulate the chemosensitivity of tumor cells [J]. Mol Cancer Ther,2008,7(1):1-9.
[35]Fujita Y K, K.Hamada, N.Ohhashi, R.Akao, Y.Nozawa, Y., Deguchi T, Ito M. Effects of miR-34a on cell growth and chemoresistance in prostate cancer PC3 cells [J]. Biochem Biophys Res Commun,2008,377(1):114-9.
[36]Xia L Z, D.Du, R.Pan, Y.Zhao, L.Sun, S.Hong, L., Liu J, Fan D. miR-15b and miR-16 modulate multidrug resistance by targeting BCL2 in human gastric cancer cells [J]. International journal of cancer Journal international du cancer,2008,123(2):372-9.
[37]To K K Z, Z.Litman, T.Bates, S. E. Regulation of ABCG2 expression at the 3' untranslated region of its mRNA through modulation of transcript stability and protein translation by a putative microRNA in the S1 colon cancer cell line [J]. Molecular and cellular biology,2008,28(17):5147-61.
[38]Yang H K, W.He, L.Zhao, J. J.O'Donnell, J. D.Wang, J.Wenham, R. M., Coppola D, Kruk P A, et al. MicroRNA expression profiling in human ovarian cancer:miR-214 induces cell survival and cisplatin resistance by targeting PTEN [J]. Cancer Res,2008, 68(2):425-33.
[39]Ahmed N A, K.Findlay, J., Quinn M. Cancerous ovarian stem cells:obscure targets for therapy but relevant to chemoresistance [J]. Journal of cellular biochemistry,2013, 114(1):21-34.
[40]Meng E L, B.Sullivan, P.McClellan, S.Finan, M. A., Reed E, Shevde L, et al. CD44+/CD24-ovarian cancer cells demonstrate cancer stem cell properties and correlate to survival [J]. Clinical & experimental metastasis,2012,29(8):939-48.
[41]Luo L Z, J.Liang, B.Zhao, Z.Sun, L., Cao D, Yang J, et al. Ovarian cancer cells with the CD117 phenotype are highly tumorigenic and are related to chemotherapy outcome [J]. Experimental and molecular pathology,2011,91(2):596-602.
[42]Han Y H, H.Xiao, Z.Zhang, W.Cao, Y.Qu, L.Shou, C. Integrated analysis of gene expression profiles associated with response of platinum/paclitaxel-based treatment in epithelial ovarian cancer [J]. PLoS One,2012,7(12):e52745.
[43]Lee J K H, D. M.Cho, H.Weinstein, J. N.Kaldjian, E. P., Karpovich J, Grimshaw A, et al. A strategy for predicting the chemosensitivity of human cancers and its application to drug discovery [J]. Proceedings of the National Academy of Sciences of the United States of America,2007,104(32):13086-91.
[2]Hernandez B Y G, M. D.Cassel, K. D.Pobutsky, A. M., Vu V, Wilkens L R. Preview of Hawaii Cancer Facts and Figures 2010 [J]. Hawaii medical journal,2010,69(9): 223-4.
[3]Bookman M A B, M. F.McGuire, W. P.Harper, P. G.Alberts, D. S.Friedlander, M., Colombo N, Fowler J M, et al. Evaluation of new platinum-based treatment regimens in advanced-stage ovarian cancer:a Phase Ⅲ Trial of the Gynecologic Cancer Intergroup [J]. Journal of clinical oncology:official journal of the American Society of Clinical Oncology,2009,27(9):1419-25.
[4]Yan X D P, L. Y.Yuan, Y.Lang, J. H., Mao N. Identification of platinum-resistance associated proteins through proteomic analysis of human ovarian cancer cells and their platinum-resistant sublines [J]. Journal of proteome research,2007,6(2):772-80.
[5]Moss S E M, R. O. The annexins [J]. Genome biology,2004,5(4):219.
[6]Mussunoor S M, G. I. The role of annexins in tumour development and progression [J]. The Journal of pathology,2008,216(2):131-40.
[7]Tait J F F, D. A.Miao, C. H.Killary, A. M.Adler, D. A., Disteche C M. Chromosomal localization of the human annexin Ⅲ (ANX3) gene [J]. Genomics,1991,10(2):441-8.
[8]Cao D L Y, D. W.Zhang, H. L.Zhu, Y, Wang Y X, Yao X D. 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]. The Prostate,2011,71(7): 700-10.
[9]Bianchi C B, S.Raimondo, F.Torsello, B.Angeloni, V., Ferrero S, Di Stefano V, 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]. The American journal of pathology,2010,176(4):1660-70.
[10]Yin J Y, 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]. Journal of cellular and molecular medicine,2012,16(2):337-48.
[11]Kollermann J S, T.Bang, H.Schwall, G. P.von Eichel-Streiber, C, Simon R, Schostak M, et al. Expression and prognostic relevance of annexin A3 in prostate cancer [J]. European urology,2008,54(6):1314-23.
[12]赵洁魏.免疫组化技术的标准化质控管理[J].青岛大学医学院学报,2009,45(2)：175-6.
[13]Han Y H, H.Xiao, Z.Zhang, W.Cao, Y.Qu, L.Shou, C. Integrated analysis of gene expression profiles associated with response of platinum/paclitaxel-based treatment in epithelial ovarian cancer [J]. PLoS One,2012,7(12):e52745.
[14]E1-Rifai W, J. StuartKim, YoungchulDuska, LindaBirrer, Michael, Levine D A, Moskaluk C, et al. Multi-Gene Expression Predictors of Single Drug Responses to Adjuvant Chemotherapy in Ovarian Carcinoma:Predicting Platinum Resistance [J]. PLoS ONE,2012,7(2):e30550.
[15]DesMarais V, Ghosh M, Eddy R, et al. Cofilin takes the lead [J]. Journal of cell science,2005,118(Pt 1):19-26.
[16]Nishimura S, Tsuda H, Kataoka F, et al. Overexpression of cofilin 1 can predict progression-free survival in patients with epithelial ovarian cancer receiving standard therapy [J]. Human pathology,2011,42(4):516-21.
[17]Riedinger J M, Bonnetain F, Basuyau J P, et al. Change in CA125 levels after the first cycle of induction chemotherapy is an independent predictor of epithelial ovarian tumour outcome [J]. Annals of oncology:official journal of the European Society for Medical Oncology/ESMO,2007,18(5):881-5.
[18]Karam A K K, B. Y. Ovarian cancer:the duplicity of CA125 measurement [J]. Nature reviews Clinical oncology,2010,7(6):335-9.
[19]Therasse P A, S. G.Eisenhauer, E. A.Wanders, J.Kaplan, R. S.Rubinstein, L., Verweij J, Van Glabbeke M, et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada [J]. Journal of the National Cancer Institute,2000,92(3):205-16.
[20]Galaal K N R, Bristow RE, Patel A, Bryant A, DickinsonHO. Cytoreductive surgery plus chemotherapy versus chemotherapy alone for recurrent epithelial ovarian cancer [J]. TheCochrane Library,2010,6):
[21]Colombo N, Gore M. Treatment of recurrent ovarian cancer relapsing 6-12 months post platinum-based chemotherapy [J]. Critical reviews in oncology/hematology,2007, 64(2):129-38.
[22]Zajchowski D A, Karlan B Y, Shawver L K. Treatment-Related Protein Biomarker Expression Differs between Primary and Recurrent Ovarian Carcinomas [J]. Molecular Cancer Therapeutics,2011,11 (2):492-502.
[23]Outcome of ATP-based tumor chemosensitivity assay directed chemotherapy in heavily pre-treated recurrent ovarian carcinoma [J].
[24]Melichar B F, P.Tomsova, M.Malirova, E. Repeat chemosensitivity of epithelial ovarian carcinoma in a BRCA1 mutation carrier to paclitaxel/platinum combination chemotherapy [J]. European journal of gynaecological oncology,2009,30(3):323-5.
[1]Hernandez B Y G, M. D.Cassel, K. D.Pobutsky, A. M., Vu V, Wilkens L R. Preview of Hawaii Cancer Facts and Figures 2010 [J]. Hawaii medical journal,2010,69(9): 223-4.
[2]Tagawa T, Robert Yen, Yun, Mortimer J. Ovarian Cancer:Opportunity for Targeted Therapy [J]. Journal of Oncology,2012,2012(1-9.
[3]McGuire W P H, W. J.Brady, M. F.Kucera, P. R.Partridge, E. E.Look, K. Y, Clarke-Pearson D L, Davidson M. Cyclophosphamide and cisplatin compared with paclitaxel and cisplatin in patients with stage Ⅲ and stage Ⅳ ovarian cancer [J]. The New England journal of medicine,1996,334(1):1-6.
[4]Ledermann J A K, R. S. Optimal treatment for relapsing ovarian cancer [J]. Annals of oncology:official journal of the European Society for Medical Oncology/ESMO, 2010,21 Supp17(vii218-22.
[5]Kartalou M E, J. M. Mechanisms of resistance to cisplatin [J]. Mutation research, 2001,478(1-2):23-43.
[6]Jandial D D F-H, S.Larson, C. A., Elliott G I, Wrasidlo W J, et al. Enhanced delivery of cisplatin to intraperitoneal ovarian carcinomas mediated by the effects of bortezomib on the human copper transporter 1 [J]. Clinical cancer research:an official journal of the American Association for Cancer Research,2009,15(2):553-60.
[7]Liu J J, Jamieson S M, Subramaniam J, et al. Neuronal expression of copper transporter 1 in rat dorsal root ganglia:association with platinum neurotoxicity [J]. Cancer Chemother Pharmacol,2009,64(4):847-56.
[8]Borst P E, R.Kool, M.Wijnholds, J. A family of drug transporters:the multidrug resistance-associated proteins [J]. Journal of the National Cancer Institute,2000,92(16): 1295-302.
[9]Liedert B M, V.Schadendorf, D.Thomale, J., Lage H. Overexpression of cMOAT (MRP2/ABCC2) is associated with decreased formation of platinum-DNA adducts and decreased G2-arrest in melanoma cells resistant to cisplatin [J]. The Journal of investigative dermatology,2003,121(1):172-6.
[10]Mangala L S Z, V.Schmandt, R.Leshane, E. S.Halder, J. B., Armaiz-Pena G N, Spannuth W A, et al. Therapeutic Targeting of ATP7B in Ovarian Carcinoma [J]. Clinical cancer research:an official journal of the American Association for Cancer Research, 2009,15(11):3770-80.
[11]Halder J K, A. A.Landen, C. N., Jr.Han, L. Y.Lutgendorf, S. K., Lin Y G, Merritt W M, et al. Focal adhesion kinase targeting using in vivo short interfering RNA delivery in neutral liposomes for ovarian carcinoma therapy [J]. Clinical cancer research:an official journal of the American Association for Cancer Research,2006,12(16):4916-24.
[12]Nakayama K K, A.Ogawa, K.Miyazaki, K.Neamati, N., Takebayashi Y. Copper-transporting P-type adenosine triphosphatase (ATP7B) as a cisplatin based chemoresistance marker in ovarian carcinoma:comparative analysis with expression of MDR1, MRP1, MRP2, LRP and BCRP [J]. International journal of cancer Journal international du cancer,2002,101(5):488-95.
[13]Lewis A D H, J. D.Wolf, C. R. Glutathione and glutathione-dependent enzymes in ovarian adenocarcinoma cell lines derived from a patient before and after the onset of drug resistance:intrinsic differences and cell cycle effects [J]. Carcinogenesis,1988,9(7): 1283-7.
[14]Kang S J, W.Kim, J. W.Park, N. H.Song, Y. S.Kim, S. C., Park S Y, Kang S B, et al. Association between excision repair cross-complementation group 1 polymorphism and clinical outcome of platinum-based chemotherapy in patients with epithelial ovarian cancer [J]. Experimental & molecular medicine,2006,38(3):320-4.
[15]Steffensen K D W, M.Jakobsen, A. The relationship of platinum resistance and ERCC1 protein expression in epithelial ovarian cancer [J]. International journal of gynecological cancer:official journal of the International Gynecological Cancer Society, 2009,19(5):820-5.
[16]Rubatt J M, Darcy K M, Tian C, et al. Pre-treatment tumor expression of ERCC1 in women with advanced stage epithelial ovarian cancer is not predictive of clinical outcomes:a Gynecologic Oncology Group study [J]. Gynecol Oncol,2012,125(2): 421-6.
[17]Brown R H, G. L.Gallagher, W. M.McIlwrath, A. J.Margison, G. P.van der Zee, A. G., Anthoney D A. hMLHl expression and cellular responses of ovarian tumour cells to treatment with cytotoxic anticancer agents [J]. Oncogene,1997,15(1):45-52.
[18]Kamal N S S, J. C.Mendiboure, J.Planchard, D.Olaussen, K. A.Rousseau, V., Popper H, Pirker R, et al. MutS homologue 2 and the long-term benefit of adjuvant chemotherapy in lung cancer [J]. Clinical cancer research:an official journal of the American Association for Cancer Research,2010,16(4):1206-15.
[19]Shachar S, Ziv O, Avkin S, et al. Two-polymerase mechanisms dictate error-free and error-prone translesion DNA synthesis in mammals [J]. The EMBO journal,2009,28(4): 383-93.
[20]Galluzzi L S, L.Vitale, I.Michels, J., Martins I, Kepp O, et al. Molecular mechanisms of cisplatin resistance [J]. Oncogene,2012,31(15):1869-83.
[21]Narod S A F, W. D. BRCA1 and BRCA2:1994 and beyond [J]. Nature reviews Cancer,2004,4(9):665-76.
[22]Gourley C M, C. O.Roxburgh, P.Yap, T. A.Harden, S., Paul J, Ragupathy K, et al. Increased incidence of visceral metastases in Scottish patients with BRCA1/2-defective ovarian cancer:an extension of the ovarian BRCAness phenotype [J]. Journal of clinical oncology:official journal of the American Society of Clinical Oncology,2010,28(15): 2505-11.
[23]Dann R B D, J. A.Timms, K. M.Zom, K. K., Potter J, Flake D D,2nd, et al. BRCA1/2 mutations and expression:response to platinum chemotherapy in patients with advanced stage epithelial ovarian cancer [J]. Gynecol Oncol,2012,125(3):677-82.
[24]Gadducci A C, S.Muraca, S.Genazzani, A. R. Molecular mechanisms of apoptosis and chemosensitivity to platinum and paclitaxel in ovarian cancer:biological data and clinical implications [J]. European journal of gynaecological oncology,2002,23(5): 390-6.
[25]Williams J L, P. C.Griffith, K. A.Choi, M.Fogoros, S., Hu Y Y, Liu J R. Expression of Bcl-xL in ovarian carcinoma is associated with chemoresistance and recurrent disease [J]. Gynecol Oncol,2005,96(2):287-95.
[26]Chen L L, L.Yan, X.Liu, N.Gong, L., Pan S, Lin F, et al. Survivin status affects prognosis and chemosensitivity in epithelial ovarian cancer [J]. International journal of gynecological cancer:official journal of the International Gynecological Cancer Society, 2013,23(2):256-63.
[27]Hu J F, E. Depleting Mirk Kinase Increases Cisplatin Toxicity in Ovarian Cancer Cells [J]. Genes & cancer,2010,1(8):803-11.
[28]Yu H S, J.Xu, Y.Kang, J., Li H, Zhang L, et al. p62/SQSTM1 involved in cisplatin resistance in human ovarian cancer cells by clearing ubiquitinated proteins [J]. European journal of cancer (Oxford, England:1990),2011,47(10):1585-94.
[29]Sherman-Baust C A W, A. T.Rangel, L. B., Pizer E S, Cho K R, et al. Remodeling of the extracellular matrix through overexpression of collagen VI contributes to cisplatin resistance in ovarian cancer cells [J]. Cancer cell,2003,3(4):377-86.
[30]Pan S C, L.White, J. T.Lu, W.Utleg, A. G., Yan X, Urban N D, et al. Quantitative proteomics analysis integrated with microarray data reveals that extracellular matrix proteins, catenins, and p53 binding protein 1 are important for chemotherapy response in ovarian cancers [J]. OMICS,2009,13(4):345-54.
[31]Bartel D P. MicroRNAs:genomics, biogenesis, mechanism, and function [J]. Cell, 2004,116(2):281-97.
[32]Ma L T-F, J.Weinberg, R. A. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer [J]. Nature,2007,449(7163):682-8.
[33]Climent J D, P.Fridlyand, J.Palacios, J.Siebert, R.Albertson, D. G., Gray J W, Pinkel D, et al. Deletion of chromosome 11q predicts response to anthracycline-based chemotherapy in early breast cancer [J]. Cancer Res,2007,67(2):818-26.
[34]Blower P E C, J. H.Verducci, J. S.Lin, S.Park, J. K.Dai, Z.Liu, C. G., Schmittgen T D, Reinhold W C, et al. MicroRNAs modulate the chemosensitivity of tumor cells [J]. Mol Cancer Ther,2008,7(1):1-9.
[35]Fujita Y K, K.Hamada, N.Ohhashi, R.Akao, Y.Nozawa, Y., Deguchi T, Ito M. Effects of miR-34a on cell growth and chemoresistance in prostate cancer PC3 cells [J]. Biochem Biophys Res Commun,2008,377(1):114-9.
[36]Xia L Z, D.Du, R.Pan, Y.Zhao, L.Sun, S.Hong, L., Liu J, Fan D. miR-15b and miR-16 modulate multidrug resistance by targeting BCL2 in human gastric cancer cells [J]. International journal of cancer Journal international du cancer,2008,123(2):372-9.
[37]To K K Z, Z.Litman, T.Bates, S. E. Regulation of ABCG2 expression at the 3' untranslated region of its mRNA through modulation of transcript stability and protein translation by a putative microRNA in the S1 colon cancer cell line [J]. Molecular and cellular biology,2008,28(17):5147-61.
[38]Yang H K, W.He, L.Zhao, J. J.O'Donnell, J. D.Wang, J.Wenham, R. M., Coppola D, Kruk P A, et al. MicroRNA expression profiling in human ovarian cancer:miR-214 induces cell survival and cisplatin resistance by targeting PTEN [J]. Cancer Res,2008, 68(2):425-33.
[39]Ahmed N A, K.Findlay, J., Quinn M. Cancerous ovarian stem cells:obscure targets for therapy but relevant to chemoresistance [J]. Journal of cellular biochemistry,2013, 114(1):21-34.
[40]Meng E L, B.Sullivan, P.McClellan, S.Finan, M. A., Reed E, Shevde L, et al. CD44+/CD24-ovarian cancer cells demonstrate cancer stem cell properties and correlate to survival [J]. Clinical & experimental metastasis,2012,29(8):939-48.
[41]Luo L Z, J.Liang, B.Zhao, Z.Sun, L., Cao D, Yang J, et al. Ovarian cancer cells with the CD117 phenotype are highly tumorigenic and are related to chemotherapy outcome [J]. Experimental and molecular pathology,2011,91(2):596-602.
[42]Han Y H, H.Xiao, Z.Zhang, W.Cao, Y.Qu, L.Shou, C. Integrated analysis of gene expression profiles associated with response of platinum/paclitaxel-based treatment in epithelial ovarian cancer [J]. PLoS One,2012,7(12):e52745.
[43]Lee J K H, D. M.Cho, H.Weinstein, J. N.Kaldjian, E. P., Karpovich J, Grimshaw A, et al. A strategy for predicting the chemosensitivity of human cancers and its application to drug discovery [J]. Proceedings of the National Academy of Sciences of the United States of America,2007,104(32):13086-91.