摘要
背景和目的：
卵巢癌是妇科肿瘤首位致死性疾病,晚期患者的5年生存率始终徘徊在20%～30%,肿瘤细胞对于化疗药物产生耐药性是造成这一状况的主要原因之一。铂类是治疗卵巢癌的一线化疗药物,临床对于卵巢癌铂类药物耐药已有明确的认识和定义,但对其耐药性产生机制的认识相对处于初级阶段,进一步预测铂类化疗耐药的工作则更是刚刚起步。
本课题组自2003年起开展对卵巢上皮癌铂类化疗耐药相关蛋白的研究,运用比较蛋白质组学技术筛选出铂类化疗耐药相关蛋白Annexin A3。经免疫组织化学验证, Annexin A3在临床卵巢癌铂类耐药患者的肿瘤组织中表达明显升高,Annexin A3高表达组患者的无瘤生存期显著缩短。动物试验表明,Annexin A3高表达细胞,在顺铂作用后裸鼠中成瘤率明显上升。运用质粒转染技术,上调Annexin A3的表达后,发现Annexin A3能够降低细胞内铂含量及铂-DNA结合量。Annexin A3高表达的细胞对铂类化疗药物的耐药性显著增强,但是对紫杉醇、表阿霉素等药物无交叉耐药性,提示Annexin A3可能是铂类特异性耐药相关蛋白。进一步的研究发现,在卵巢上皮癌细胞系的培养基上清中有Annexin A3蛋白表达,其强度与细胞内Annexin A3的表达水平呈正相关。体内实验在临床卵巢癌患者的外周血中也检测出Annexin A3蛋白的表达,充分证实Annexin A3是一个可分泌蛋白。
本研究旨在前期工作的基础上探讨利用ELISA法在外周血定量检测AnnexinA3蛋白的可行性。分析Annexin A3蛋白在正常妇女、卵巢良性肿瘤患者及卵巢癌患者外周血中的分布及表达水平,并初步探讨卵巢癌患者外周血表达水平与卵巢癌铂类耐药的相关性。
方法：
1.研究对象：连续入选于2009年12月至2010年2月期间在北京协和医院体检及就诊的健康妇女、卵巢良性肿瘤和卵巢癌患者。
健康妇女入选标准：纳入妇科体格检查、宫颈细胞学检查及妇科超声检查均无异常,并同时进行了血清CA125检测的妇女,排除既往有卵巢肿瘤或妇科及全身恶性肿瘤的妇女。年龄与卵巢癌患者匹配。
卵巢良性肿瘤入选标准：在北京协和医院住院手术治疗的患者,病理诊断明确,术前进行了血清CA125检测。
卵巢癌患者入选标准：在北京协和医院妇产科治疗及随诊,病理诊断为卵巢原发上皮癌,进行了血清CA125的检测,排除合并其它妇科或全身恶性肿瘤的患者。卵巢癌耐药病例包括耐药性和难治性卵巢癌。耐药性卵巢癌为,初次化疗有效,但停化疗6个月以内肿瘤复发。难治性卵巢癌为,初次化疗时疾病进展或持续存在。将二者作为铂耐药性卵巢癌。
2.标本及临床资料收集：全部标本来自北京协和医院进行血清CA125检测的剩余血清,检测后于4℃冷库内保存。7天之内将血清样本分装到0.5mL Eppendorf中,转移至-80℃冰箱冻存。在测量前半小时一次化冻。详细记录同一样本的血清CA125水平以及所有对象相应的临床资料。
3.采用ELISA方法检测上述样本外周血清Annexin A3的浓度。每份样本设3个复孔,取均值求得测定值。对于检测过程的质量控制措施包括：制定标准的操作程序,培训操作人员,一次性完成测定,在临床检验专家的指导下设置标准品和标准曲线,随机插入已知蛋白浓度的质控品,被检样品的随机上样,以及采用双波长测定吸收值等。
4.采用回顾性分析的方法,分层分析临床卵巢上皮癌化疗敏感和耐药病例的各生物学变量与Annexin A3蛋白表达水平的相关性；分析卵巢上皮癌患者的无铂化疗间期与Annexin A3蛋白表达水平的相关性。
5.采用SPSS17.0软件进行统计学分析。利用Crubbs氏法对ELISA测定进行质控检验。Kolmogorov-Smimov非参数检验进行质控品光密度及样本的正态性检验。两组正态分布独立样本比较采用t检验。两组非正态分布独立样本比较利用Mann-Whitney非参数检验。多组非正态分布独立样本比较利用Kruskal-Wallis非参数检验。
研究结果：
1.连续收集病例共计271例。其中,健康妇女102例,年龄范围31—78岁,中位年龄52岁。卵巢良性肿瘤患者47例,年龄范围17—61岁,中位年龄36岁。卵巢癌患者122例,包括化疗敏感病人63例,耐药病人59例；年龄范围31—81岁,中位年龄53岁。卵巢癌患者与健康妇女的年龄无显著性差异；与卵巢良性肿瘤患者的年龄有显著性差异(p<0.05)。
2.271份标本共使用10个96孔板。经Crubbs氏法质控检验,第1～9板符合质控要求,第10板不符合质控要求。舍弃板10数据,共计26份标本,包括卵巢良性肿瘤8例以及健康妇女18例。Kolmogorov-Smirnov检验对质控品光密度进行正态性检验,三个浓度p值分别为0.919,0.751,0.931,均符合正态分布。并去除标准曲线以外的极端大值6例和极端小值8例。
3.在临床全部271份的血清样本中均可以检测到Annexin A3蛋白的表达。去掉极端值及质控不合格值后,共有样本231份进行统计分析。经Kolmogorov-Smimov检验,卵巢良性肿瘤患者Annexin A3测定值为正态分布,p=0.387。健康妇女和卵巢癌患者Annexin A3非正态分布,p值分别为0.028,0.007。在健康妇女血清中的
Annexin A3表达范围在0.261-4.74ng/mL,平均值0.867ng/mL,中位数0.709ng/mL。卵巢良性肿瘤患者Annexin A3蛋白的表达范围在0.263-2.46ng/mL,平均值0.815ng/mL,中位数0.740ng/mL。卵巢癌患者Annexin A3蛋白的表达范围在0.201-2.84ng/mL,平均值0.689ng/mL,中位数0.567ng/mL。经Kruskal-Wallis检验,三组相比无显著性差异。分析研究对象血清Annexin A3的表达水平与CA125测定值的相关性。将健康妇女血清CA125值分为<10U/mL和≥10U/mL两组,Annexin A3的表达水平无明显差别。将卵巢良性肿瘤患者血清CA125值分为<35U/mL和≥35U/mL两组,Annexin A3的表达水平也无明显差别。将卵巢癌患者血清CA125值分为<100U/mL和≥100U/mL两组,前者的Annexin A3的测定平均值为0.645ng/mL,后者平均值为0.840ng/mL,两组的表达水平有显著性差别(p=0.043)。
4.根据卵巢上皮癌患者末次化疗的时间将研究对象分为化疗敏感和耐药两组。按照患者的年龄、血清CA125水平、FIGO分期、肿瘤的病理学类型、分化程度、以及铂化疗的疗程总数等生物学变量进行分层,两组之间Annexin A3蛋白的表达水平均未达到统计学上的显著性差别。进一步根据卵巢癌患者末次铂类化疗的时间统计无铂化疗间期,分为≤6个月和>6个月两组,按照Annexin A3的表达水平的测定值分层,取所采用试剂盒建议的标曲范围最小值0.8ng/mL为上限,以0.2ng/mL为一个层间距,至试剂盒的最小分辨度为0.2ng/mL为下限,共分为4层。分析表明,伴随分层Annexin A3基础数值的递增,两组Annexin A3测定值的的平均值和中位数平行线性上升。至Annexin A3≥0.6ng/mL层时,两个组Annexin A3表达水平同步进入正态分布区间,采用t检验对两组间的平均值进行比较。在Annexin A3≥0.6ng/mL层,Annexin A3的均值,无铂化疗间期≤6个月组为1.17±0.615ng/mL;>6个月组为0.924±0.250ng/mL, p=0.048。在Anneixn A3≥0.8ng/mL层,AnnexinA3的均值,无铂化疗间隔≤6个月组为1.44±0.647ng/mL；>6个月组水平为1.07±0.214ng/mL, p=0.028。由于两组的方差齐性检验p值均<0.05,进一步采用t’检验,获得对Annexin A3均值检验的p值,在Annexin A3≥0.6ng/mL层,p=0.095；在Annexin A3≥0.8ng/mL层,p=0.070；统计学十分接近显著性差别。
结论：
1.本研究首次连续收集较大量的临床标本开展卵巢癌铂类耐药相关蛋白Annexin A3在人外周血中检测的研究。共收集健康妇女102例,卵巢良性肿瘤患者47例,卵巢癌患者122例,共计271例研究对象。
2.采用ELISA法可以完成对Annexin A3蛋白在人外周血清中表达的高通量定量检测。在临床全部271份的血清样本中均检测到Annexin A3蛋白的表达,健康妇女和卵巢癌患者Annexin A3非正态分布。有超过150例标本的测定值低于建议标曲范围的最小值0.8ng/mL,表明现有ELISA试剂盒的敏感性欠佳。开发更加敏感的检测方法可以完善对Annexin A3蛋白在人外周血中检测的研究。
3.现有样本中,Annexin A3蛋白在健康女性、卵巢良性肿瘤患者和卵巢上皮癌患者外周血清中的表达无显著性差异。Annexin A3蛋白的表达与健康女性和卵巢良性肿瘤患者外周血中CA125水平无明显相关性；而与卵巢上皮癌患者外周血中高水平CA125的表达有关,提示Annexin A3蛋白可能是卵巢癌特异性相关蛋白。本研究中卵巢癌患者Annexin A3蛋白的测定值在直观上低于健康女性和卵巢良性肿瘤患者,可能与前者均为术后化疗或化疗后随访的病例,其肿瘤负荷大大降低有关。检测卵巢癌患者术前或化疗前血清Annexin A3蛋白水平将有助于证明Annexin A3蛋白能否成为卵巢癌铂类耐药标记蛋白。
4.本研究表明,根据卵巢上皮癌患者末次化疗的时间将研究对象分为化疗敏感和耐药两组,按照患者的年龄、血清CA125水平、FIGO分期、肿瘤的病理学类型、分化程度、以及铂化疗的疗程总数等生物学变量进行分层,两组之间Annexin A3蛋白的表达水平均未达到统计学上的显著性差别。根据卵巢癌患者末次铂类化疗的时间统计无铂化疗间期,分为≤6个月和>6个月两组,按照Annexin A3的表达水平的测定值分层,结果表明,在Annexin A3≥0.6ng/mL层时,其表达水平在≤6个月组升高,统计学十分接近显著性差别。提示Annexin A3极有可能是一种卵巢癌铂类耐药特异性相关蛋白,可以通过增加样本的数量完善研究结论。
Backgroud and Purpose:
Epithelial ovarian cancer (EOC) is the leading cause of death among the gynecological cancers with5-years survival around20%to30%in the patients with advanced disease. Resistance of tumor cell to chemotherapeutic drugs is one of the main causes of treatment failure. Platinum is the first-line drug against epithelial ovarian cancer, and there is a clear explanation and definition of clinical platinum-resistance. But understanding of its mechanism is relatively in the initial stage and predicting the platinum-resistance is still in its infancy actually.
Since2003, our research team has studied on platinum-resistance related proteins in EOC. The platinum-resistance related protein Annexin A3was selected by comparative proteomics. Validation of Annexin A3by immunohistochemistry identified its significantly high expression in tumor tissues of platinum-resistant patients, whose disease-free survival was much shorter. In animal experiments, when cisplatin treatment was administrated to nude mice xenografted with Annexin A3overpression tumors, the inhibition of tumor growth was significantly reduced. After transfecting Annexin A3-expressing plasmid into drug sensitive cells, the intracellular concentration of platinum and platinum-DNA binding capacity lowered. The Annexin A3overexpression cells showed increase in resistance to platinum compound, while there was no cross-resistance to paclitaxel, epirubicin or other drugs. It suggested that Annexin A3might be the specific platinum-resistant protein. It was found that Annexin A3could be detected in culture medium supernatant of ovarian cancer cell lines, and its concentration was positively correlated with its intra-cellular levels. In vivo, Annexin A3was also detected in serum of ovarian cancer patients, which demonstrated that Annexin A3was a secreted protein.
The aims of this research were exploring the feasibility of quantitative detection of Annexin A3protein in the serum by ELISA method on the basis of previous work, analyzing the distribution and expression of serum Annexin A3protein in healthy women, benign ovarian tumor patients and ovarian cancer patients and studying the correlation between the platinum-resistance and serum Annexin A3level of ovarian cancer patients.
Methods:
1. Objectives:the healthy women, the benign ovarian tumor patients and the ovarian cancer patients were continuously enrolled in Peking Union Medical College Hospital from Dec2009to Feb2010.
Inclusion criteria of healthy women included normal gynecological physical examination, cervical cytology and gynecological ultrasonography. Additional entry criteria included serum CA125assay. Women with gynecological or systemic malignancies were excluded. Age distribution was matched with ovarian cancer patients.
Inclusion criteria of benign ovarian tumor patients included inpatients who were treated surgically with definite pathological diagnosis. Additional entry criteria included serum CA125assay before the operation.
Inclusion criteria of ovarian cancer patients included epithelial ovarian cancer patients who were treated and followed up in Peking Union Medical College Hospital. Patients with other gynecological or systemic malignancies were excluded. Platinum-resistant disease was defined as occurrence of disease progression within6months after completing therapy or disease progression during treatment with platinum-based chemotherapy.
2. Collecting the specimens and clinical records:all serum specimens were from the remainder of clinical serum CA125assay, which were kept at4℃refrigerator for no more than7days. Then serum samples were packed in several0.5mL Eppendorfs and transferred to-80℃refrigerator. All the samples were unfrozen only once just before the ELISA assay. The CA125levels of the same serum samples and the corresponding clinical data were recorded.
3. Detecting the serum concentration of Annexin A3by ELISA kit. Each sample was set with3duplicates. Mean of the three measured samples was the actual value. The quality control process include:formulating the Standard Operative Procedures (SOP), training operators, carrying out measurement at one time, setting the standard products and standard curve under the guidance of clinical laboratory experts, utilizing quality control products whose concentration were already known, and measuring by dual-wave length absorbance.
4. All the clinical data were analyzed retrospectively. The correlations between Annexin A3level and the other clinical variables in platinum sensitive and resistant cases were stratified analyzed. The correlation between the Annexin A3level and platinum-free interval of ovarian cancer patients were determined.
5. SPSS17.0software was utilized for statistical analysis. The Crubbs' method was employed for the quality control test of ELISA. The Kolmogorov-Smirnov test was adopted to test the normal distribution of the quality control samples and others. The t test was employed to compare the means of two independent-samples with normal distribution. The Mann-Whitney nonparametric test was employed to compare the distrbution of two independent-samples with unknown distribution. The Kruskal-Wallis nonparametric test was employed to compare the distribution of multiple independent-samples with unknown distribution.
Results:
1.271cases were continuously enrolled, including102healthy women (age range:31-78yo, median age:52yo),47benign ovarian tumor patients (age range:17-61yo, median age:36yo) and122ovarian cancer patients (age range:31-81yo, median age:53yo), and the last consisted of63platinum-sensitive patients and59platinum-resistant patients. Age distributions of ovarian cancer patients and healthy women was not significantly different, whereas ones of ovarian cancer patients and benign ovarian tumor patients was significantly different (p<0.05).
2.10plates (96-well) were used for assay of total271specimens. Quality control was tested by Crubbs' method. Quality control was satisfactory in plates1to9, whereas the quality control of plate10did not meet requirements. Total26values from8benign ovarian tumor patients and18healthy women in plate10samples were excluded. The Kolmogorov-Smirnov test of quality control samples showed normal distribution of Annexin A3in three groups (p=0.919,0.751and0.931respectively).6cases of extremely high value and8cases of extremely low value, which exceeded standard curve limits, were excluded.
3. Annexin A3protein could be detected in all271samples. After removing extreme values,231samples were analyzed totally. By Kolmogorov-Smirnov test, Annexin A3level distribution in benign ovarian tumor patients showed normal distribution (p=0.387), in healthy women and ovarian cancer patients showed non-normal distribution (p=0.028and0.007, respectively). Serum Annexin A3level in healthy women ranged0.261-4.74ng/mL (mean:0.867ng/mL, median:0.709ng/mL), in benign ovarian tumor patients ranged0.263-2.46ng/mL (mean:0.815ng/mL, median:0.740ng/mL), in ovarian cancer patients ranged0.201-2.84ng/mL (mean:0.689ng/mL, median:0.567ng/mL). By Kruskal-Wallis test, there was no significant difference among these three groups. The correlation between Annexin A3expression and CA125level was analyzed. Healthy women were divided into two groups according to serum CA125level (<10U/mL and≥10U/mL), Annexin A3expression was not significantly different between these two groups. Also, Annexin A3expression was not significantly different neither between two groups from benign ovarian tumor patients (serum CA125<35U/mL and CA125> 35U/mL). Whereas among ovarian cancer patients, Annexin A3concentration was significantly higher in the patients whose serum CA125≥100U/mL than the patients whose serum CA125<100U/mL (p=0.043). The means of these two groups were0.840ng/mL and0.645ng/mL respectively.
4. Epithelial ovarian cancer patients were divided into platinum-sensitive and platinum-resistant groups based on disease-free interval. There was no significant difference of Annexin A3levels between the two groups in the stratified analysis which were stratified by the age, FIGO staging, pathological types, tumor differentiation and the total number of the platinum-based chemotherapy cycles. Furthermore, ovarian cancer patients were divided into two groups according to platinum-free interval (PFI<6months and PFI>6months). Stratified analysis was employed based on Annexin A3level. The most upper stratum was≥0.8ng/mL which was recommended as the lowest boundary of the standard curve by the kit instructions, and the lower limit of stratum was≥0.2ng/mL which was the lowest concentration the kit could distinguish. The interval of the strata was0.2ng/mL. Analysis showed that the mean and median of Annexin A3of each group increased simultaneously in accord with the increasing Annexin A3level stratum. When Annexin A3level≥0.6ng/mL, the expressions of Annexin A3in two groups were both normal distribution, so that t test was employed to compare the means between two groups. In Annexin A3≥0.6ng/mL stratum, the mean of Annexin A3level in PFI≤6months group was1.17±0.615ng/mL, while in PFI>6months group was0.924±0.250ng/mL(p=0.048). In Annexin A3≥0.8ng/mL stratum, the mean of Annexin A3level in PFI≤6months group was1.44±0.647ng/mL, while that in PFI>6months group was1.07±0.214ng/mL(p=0.028). Since homogeneity of variance in both groups were not accepted (p<0.05in both groups), so t'test was adopted instead to compare the means. In Annexin A3>0.6ng/mL stratum, p=0.095; in the Annexin A3≥0.8ng/mL stratum, p=0.070. It nearly reached the statistical significance.
Conclusions:
1.For the first time, this study continuously collected large number of clinical specimens of human peripheral serum and measured platinum-resistant related protein Annexin A3level.271cases were studied totally, including102healthy women,47benign ovarian tumor patients and122ovarian cancer patients.
2. Annexin A3expressions in human peripheral blood could be detected by quantitative high-throughput ELISA method. Annexin A3protein had been measured in all271specimens. The distributions of Annexin A3level in healthy women and ovarian cancer patients were non-normal distribution. More than150measured values were below 0.8ng/mL (which was recommended by the Elisa kit as lowest limit of standard curve), suggesting that the sensitivity of ELISA kit available was not satisfactory. Development of more sensitive assay can improve on measurement of Annexin A3protein in human peripheral blood.
3. In this study, there was no significantly different of Annexin A3levels in peripheral blood among healthy women, benign ovarian tumor patients and ovarian cancer patients. Annexin A3and serum CA125levels in healthy women and benign ovarian tumor patients were not significantly correlated. In contrast, Annexin A3expression significantly correlated with high level CA125in ovarian cancer patients, suggesting that Annexin A3protein might be ovarian cancer specific protein. In the result, it seemed that Annexin A3level in ovarian cancer patients was lower than that in healthy women and benign ovarian tumor patients. That may be the result of lower tumor volumn after the surgery and chemotherapy. Measurement of serum Annexin A3in ovarian cancer patients before surgery and chemotherapy might identify whether Annexin A3is a platinum-resistant biomarker.
4. Epithelial ovarian cancer patients were divided into platinum-sensitive and platinum-resistant groups based on disease-free interval. There was no significant difference of Annexin A3level between the two groups in the stratified analysis which was stratified by the age, FIGO staging, CA125level, pathological types, tumor differentiation and the total number of the platinum-based chemotherapy cycles. Furthermore, ovarian cancer patients were divided into two groups based on platinum-free interval (PFI<6months and PFI>6months). Stratified analysis was employed based on Annexin A3level. When Annexin A3≥0.6ng/mL, Annexin A3level in former group was higher than that in the latter group. It nearly reached the statistical significance. It highly suggested that Annexin A3is an ovarian cancer-specific platinum-resistant protein, and the conclusions could be improved by increasing the number of samples enrolled in the study.
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