摘要
研究背景与目的
卵巢恶性肿瘤是妇科三大恶性肿瘤之一,死亡率高居妇科肿瘤之首,是严重威胁妇女生命和健康的主要妇科肿瘤。尽管手术技巧的提高和新型化疗药物的问世改善了晚期卵巢恶性肿瘤患者的近期生活质量,但5年生存率仍徘徊在30%左右。造成这一困境的主要原因就是由于目前临床上尚缺乏便捷而有效的常规筛查手段和早期诊断策略,多数患者就诊时已错失手术治疗的最佳时期。因此,早期诊断是改善卵巢恶性肿瘤患者预后的关键所在。但仅仅依靠常规妇科盆腔检查远远不能满足早期诊断的需要,因为在疾病早期肿瘤仅局限于卵巢时诊断非常困难。所以寻找有实用价值的肿瘤标志物一直是妇科肿瘤研究领域的重要课题。
肿瘤标志物的检测不仅可用于恶性肿瘤的诊断,还可以用于疗效评估和治疗后病情的监测及复发癌的早期发现。甲胎蛋白(alpha-fetoprotein, AFP)和人绒毛膜促性腺激素(human chorionic gonadotropin, hCG)对内胚窦瘤和绒毛膜癌的诊断价值是不言而喻的。但对大多数卵巢恶性肿瘤而言,至今尚未找到特异性和敏感性堪与AFP和hCG相媲美的肿瘤标志物。目前,癌抗原125 (cancer antigen 125, CA125)是已发现的最重要的卵巢癌相关抗原,也是全世界范围内应用最广泛的卵巢上皮性肿瘤标志物,对卵巢浆液性囊腺癌诊断的准确率在80%以上。CA125对于卵巢上皮性癌的诊断价值已基本得到公认。但是由于部分卵巢非上皮性恶性肿瘤患者的血清CA125并不升高,而一些非恶性疾病患者的血清CA125也可升高,致使CA125仍不能单独作为诊断卵巢恶性肿瘤的指标,其临床应用受到局限。因此,探索CA125与其他肿瘤标志物联合检测以提高诊断的敏感性和特异性已成为当下临床工作中面临的巨大挑战之一。
从分子水平探讨卵巢恶性肿瘤的发病机制,是近年来妇科肿瘤研究中的热点,也是发现新的肿瘤标志物的切入点。研究表明,表遗传学机制(epigenetic mechanism)参与了恶性肿瘤的形成和发展过程,在基因转录的调控过程中起着极其重要的作用。表遗传学调控的分子机制包括DNA甲基化(DNAmethyaltion)、组蛋白修饰(histone modification)、染色体重塑(chromatin remodeling)和小RNA (microRNA)调控等。大量研究表明,基因启动子区域CpG岛(promoter region CpG islands)的异常甲基化是抑癌基因(tumor suppressor gene, TS G)和其他肿瘤相关基因沉寂(silence)失活的重要原因,有时是TSG失活的唯一原因。因此,基因启动子区域CpG岛的甲基化(简称基因启动子甲基化)有望成为某些恶性肿瘤的分子标志物。
SLIT2 (slit homolog 2 (Drosophila))基因最早在果蝇体内发现,其编码的蛋白质Slit2参与中枢神经系统的分化和发育,对轴突生长和神经元迁移有导向作用。近来有报道Slit2蛋白在乳腺癌、小细胞肺癌、结肠癌、神经胶质瘤中表达异常。
3-OST-2 (heparan sulfate D-glucosaminyl 3-O-sulfotransferase 2)即硫酸乙酰肝素D-氨基葡萄糖-3-O磺基转移酶2基因,其编码的磺基转移酶参与硫酸肝素蛋白聚糖分子中粘多糖链的最后修饰过程。此修饰过程对决定硫酸肝素蛋白聚糖与多种蛋白结合的特异性以及其调节特性具有重要意义。研究发现,3-OST-2基因启动子甲基化后造成的转录沉默表明了硫酸肝素修饰的改变参与了肿瘤的发生与发展过程。
CDH13 (cadherin 13)即H-Cadherin基因,其编码的钙依赖性细胞粘附糖蛋白是心肌细胞间相互作用的重要媒介,是神经细胞生长的负性调节因子。研究发现,CDH13蛋白在结肠癌、食道癌、胰腺癌、肺癌、宫颈癌、子宫内膜癌和何杰金淋巴瘤中表达下调,且与CDH13基因启动子的甲基化有关。
TES (testis derived transcript)即睾丸素基因,含有3个富含半胱氨酸的区域(LIM),编码一种分子量较小的细胞骨架联合蛋白,通过与其他细胞骨架蛋白相互结合而发生作用,参与细胞的粘附、细胞的扩散和肌动蛋白细胞骨架的改建,并可抑制肿瘤细胞的生长。
上述4种基因作为候选(candidate)的抑癌基因,其启动子区域CpG岛的甲基化状况已在结直肠癌、肺癌、乳腺癌、肝癌、胶质瘤及淋巴细胞白血病等肿瘤中进行了较为深入的研究,结果表明这些基因启动子的甲基化与肿瘤的发生发展有关,但鲜见其在卵巢恶性肿瘤中的甲基化状况及与卵巢恶性肿瘤发生发展关系的报道。
甲基化特异的聚合酶链反应(Methylation specific PCR, MSP)是目前研究甲基化最敏感的实验技术之一,能发现0.1%的甲基化DNA(-50pg)。其基本原理为：单链DNA经过亚硫酸氢钠修饰后,所有未甲基化的胞嘧啶(cytosine, C)脱氨转变为尿嘧啶(uracil, U),而CpG二核苷酸中的胞嘧啶则因甲基化而保持不变。据此,分别设计两对针对甲基化和非甲基化等位基因的引物,通过PCR扩增即可将甲基化与非甲基化DNA序列区分开来。
本研究采用MSP对以上4种候选的TSGs在卵巢恶性肿瘤患者的组织、外周血和腹水中的甲基化状况进行检测,以探讨DNA甲基化作为肿瘤分子标志在卵巢恶性肿瘤的诊断和早期诊断中的可行性。
本研究分为以下三部分：
第一部分卵巢恶性肿瘤组织中SLIT2、3-OST-2、CDH13和TES基因启动子甲基化的检测及临床意义
目的：探讨抑癌基因启动子的甲基化作为卵巢恶性肿瘤诊断和早期诊断分子标志物的可行性。
材料与方法：采用MSP对79例卵巢恶性肿瘤、40例卵巢良性肿瘤组织标本中SLIT2、3-OST-2、CDH13和TES基因启动子的甲基化状况进行检测,并分析甲基化与临床病理特征之间的关系。
结果：1.卵巢恶性肿瘤组织中,SLIT2、3-OST-2、CDH13和TES基因启动子的甲基化率分别是86.1%(68／79),77.2%(61／79),43.0%(34／79)和30.4%(24／79),至少有一个基因发生甲基化的频率为97.5%(77／79),卵巢良性肿瘤中则未检测到任何一种基因的甲基化。2.甲基化与肿瘤的组织学类型、病理分级、临床病理分期和患者的年龄无相关性(P值均>0.05)；平均甲基化基因个数与肿瘤分期无相关性(P=0.195)。3.SLIT2的甲基化与淋巴结转移相关(P=0.027),CDH13的甲基化与血清CA125水平相关(P=0.034)。4.早期(Ⅰ—Ⅱ期)卵巢恶性肿瘤组织中至少有上述一个基因发生甲基化的频率为96.9%(31／32)。
结论：抑癌基因启动子的甲基化是卵巢恶性肿瘤发生中的早期和普遍事件,是卵巢恶性肿瘤早期诊断的可靠指标,多种基因联合检测,可提高诊断的准确性。
第二部分：循环DNA中SLIT2、3-OST-2、CDH13和TES基因启动子的甲基化用于卵巢恶性肿瘤早期诊断的可行性研究
目的：探讨循环DNA用于卵巢恶性肿瘤诊断和早期诊断的可行性。
材料与方法：采用MSP对42例可疑卵巢恶性肿瘤患者和30例健康女性的外周血浆标本中SLIT2、3-OST-2、CDH13和TES基因启动子的甲基化状况进行检测,并将结果与相对应的肿瘤组织中同一基因的甲基化状况进行对比、分析。
结果：1.肿瘤患者的血浆中,SLIT2、3-OST-2、CDH13和TES基因启动子的甲基化率分别是38.1%(16／42),28.6%(12／42),19.0%(8／42)和19.0%(8／42),至少一个基因发生甲基化的频率为54.8%(23／42),30例健康对照者的血浆中未检测到任一基因的甲基化。2.在由病理确诊的26例卵巢恶性肿瘤患者中,23例在外周血中检测到了至少一种基因的甲基化,敏感性为88.5%(23／26),25例在肿瘤组织中检测到了至少一种基因的甲基化,敏感性为96.2%(25／26),二者的特异性均为100%(17／17)。3.血浆中各基因甲基化的检出率与自身肿瘤组织中的甲基化率比较,差异无统计学意义(P值均>0.05),二者的一致性为95.2%(40／42)。4.若用血浆代替组织进行甲基化检测,其敏感性为92.0%(23／25),特异性为100%(17／17),准确性为95.2%(40／42),阳性预测值为100%(23／23),阴性预测值为89.5%(17／19)。5.早期(Ⅰ—Ⅱ期)恶性肿瘤患者的血浆中任一基因的甲基化率为70%(7／10),若用血浆代替组织进行甲基化检测的敏感性为77.8%(7／9),准确性为80.0%(8／10),阳性预测值为100%(7／7),阴性预测值为33.3%(1／3)。6.组织DNA中CDH13的甲基化率与CA125水平相关(P=0.021)。
结论：循环DNA中抑癌基因启动子的甲基化是卵巢恶性肿瘤的可靠标志,外周血可替代肿瘤组织用于卵巢恶性肿瘤的诊断和早期诊断。
第三部分：腹水或腹腔冲洗液中SLIT2、3-OST-2、CDH13和TES基因启动子的甲基化分析对卵巢恶性肿瘤诊断的价值
目的：探讨腹水或腹腔冲洗液用于卵巢恶性肿瘤诊断和早期诊断的价值。
材料与方法：采用MSP对42例可疑卵巢恶性肿瘤患者的腹水或腹腔冲洗液中SLIT2、3-OST-2、CDH13和TES基因启动子的甲基化状况进行检测,并将结果与腹水细胞学检查及第二部分研究结果进行对比、分析。
结果：1.患者的腹水或腹腔冲洗液中,SLTT2、3-OST-2、CDH13和TES基因启动子的甲基化率分别是47.6%(20／42),38.1%(16／42),21.4%(9／42)和23.8%(10／42),至少一个基因发生甲基化的频率为52.4%(22／42),与血浆中的甲基化率比较,差异无统计学意义(P值均>0.05)。2.在经病理检查确诊的26例恶性肿瘤患者当中,有22例在腹水或腹腔冲洗液中检测到至少一种基因的甲基化,敏感性为84.6%(22／26),特异性为100%(17／17),与血浆用于诊断的敏感性(88.5%)比较,差异无统计学意义(校正χ2检验,P=1.0)。若以腹水替代组织进行甲基化检测,其敏感性为88.0%(22／25),特异性为100%(17／17),准确性为92.9%(39／42),阳性预测值为100%(22／22),阴性预测值为85.0%(17／20),各值与循环DNA中的数值相比,差异无统计学意义。3.在20例细胞学检查结果阳性的患者当中,19例检测到了任一基因的甲基化,敏感性为95.0%(19／20),特异性为100%(22／22)。4.在16例良性肿瘤患者的腹水或腹腔冲洗液中,无一例检测到任一基因的甲基化,与血浆的检测结果一致。5.早期恶性肿瘤患者的腹腔冲洗液或腹水中任一基因的甲基化率为60%(6／10),与血浆中的甲基化率(70%)无异,但若两者同时检测,敏感性略可提高(80%,8／10)。
结论：腹水或腹腔冲洗液可替代血浆或肿瘤组织用于癌相关基因启动子的甲基化分析,对早期患者的腹水和血浆伺时进行甲基化检测,可提高诊断的准确性。
Backgroud
Ovarian carcinoma is the third most common gynecological malignancy and is the leading cause of death among women with gynecologic cancers. The lifetime risk of ovarian carcinoma is about 1 in 55, while the prevalence in postmenopausal women is 1 in 2,500, and the age-adjusted incidence is around 11 per 100,000. It is estimated that 82,550 new cases will be diagnosed, and 53,600 deaths from ovarian carcinoma occur, in Europe and America in 2009. About 190,000 new cases and 114,000 deaths from ovarian cancer are estimated to occur annually in the world. Unfortunately, there is no reliable data at the national level on mortality from ovarian carcinoma in China.
Because there is currently no sufficiently accurate screening test proven to be effective in the early detection of ovarian carcinoma,70%～75%of patients are not diagnosed until the disease has advanced to stage III or IV. The 5-year survival rate for women with these advanced stages is less than 30%, but patients with stage I disease have 5-year survival in access of 85%if appropriately treated. Because of the association between prognosis and stage, a specific and sensitive screening test is intuitively appealing. Detection of a large fraction of ovarian carcinoma in early stage would be of clearly clinical benefit and might significantly improve the overall survival. It is estimated that if the percentage of cases diagnosed in stage I could increase to 75%by early detection, mortality could be reduced by 50%, and the 5-year survival could rise to 80-95%.
Both genetic and epigenetic changes that initiate and drive tumorigenesis are promising target for early detection because they may precede clinically obvious disease. Epigenetic alterations, defined as heritable changes in gene expression but without DNA sequence changing, such as DNA methylation, histone modification, microRNAs and chromatin remodeling, are increasingly focused on recently, for their reversible nature. DNA methylation, the best studied epigenetic mechanism, is an enzymatic process to add the methyl group at the fifth carbon of cytosines within the palindromic dinucleotide 5'-CpG-3'sequence. Cytosine methylation is important for epigenetic regulation of endogenous genes, silencing of transposons and controlling of genome stability. Now, DNA methyaltion is considered a third pathway to satisfy Knudson's hypothesis. It can lead to transcriptional inactivation and gene silence by directly blocking the binding of transcriptional factors to DNA, and/or by MBP(methyl-CpG-binding protein), which recruits chromatin remodeling corepressor complexes.
Numerous studies have revealed that the coexistence of gene-specific promoter hypermethylation and global genomic DNA hypomethylation is an epigenetic characteristics of cancer cells, and the methylation of CpG dinucleotides in the promoter regions of tumor suppressor genes (TSGs) can be used as a useful biomarker for early detection of cancer. However, these analyses usually are performed after surgery or biopsy, which limits their use for early screening for cancer. Fortunately, several recent studies have demonstrated the feasibility of detecting tumor-specific genetic or epigenetic alterations in body fluids, especially in serum or plasma. Tumor-associated DNA in blood originate from cell lysis, apoptosis, necrosis or active release from the primary tumor, and has almost the same characteristics as primary tumor DNA, such as oncogene expression, tumor suppressor gene mutations, as well as microsatellite and epigenetic alterations.
Methylation-specific PCR(MSP), a sensitive assay that detects CpG dinucleotides in a CpG island, can identify one allele in the presence of 1,000～10,000 unmethylated alleles, and does not require prior knowledge of epigenetic alterations. Due to both its high sensitivity and specificity, MSP analysis provides a straightforward answer to cancer detection. To determine the feasibility of DNA methylation for early detection of ovarian carcinoma, we first investigated the methylation status of the human homologue of the Drosophila Slit2(SLIT2), Heparan sulfate D-glucosaminyl 3-O-sulfotransferase-2(3-OST-2), H-cadherin (CDH13) and TESTIN(TES) genes in tumor DNA from 79 ovarian carcinoma patients. Then we analysed the methylation status of plasma and ascites DNA from another 42 patients with a probable ovarian carcinoma diagnosis, in order to determine the sensitivity and specificity of DNA methylation for detection of cancer.
Our research has three parts and the abstrct was as followed:
Part 1:The Frequency of Hypermethylated SLIT2,3-OST-2,CDH13 and TES in Primary Ovarian Carcinoma
Purpose:To determine the feasibility of DNA methylation for early diagnosis of ovarian carcinoma.
Materials and Methods:Using Methylation-specific PCR, we first assessed the methylation status of SLIT2,3-OST-2, CDH13 and TES in tumor DNA from 79 patients with ovarian carcinoma, and analyzed the relationship between methylation status and clinicopathological parameters.
Results:
1. In the samples from primary ovarian carcinomas, the frequency of aberrant promoter hypermethylation of SLIT2,3-OST-2, CDH13 and TES genes was 86.1% (68/79),77.2%(61/79),43.0%(34/79) and 30.4%(24/79), respectively. Overall, in 77 of the 79 tumors, at least one of the four gene promoters was hypermethylated, which provides 97.5%diagnostic coverage. In contrast, none of the 40 benign ovarian tissues exhibited abnormal promoter hypermethylation of any gene.
2. Hypermethylation was detected in patients of all ages, in all histological cell types, in all pathological grades, in all stages of ovarian carcinoma, and even in borderline tumors of low malignant potential. The methylation status had no correlation with tumor stage, histological classifications, grade or patient age. There was no association between the mean number of methylated genes and the tumor stage (2.59 0.91 in stageⅠ-Ⅱvs 2.28±0.96 in stageⅢ-Ⅳ, P= 0.195).
3. Hypermethylation of CDH13 was associated with CA 125 level (P= 0.034) and SLIT2 was associated with the lymph node metastasis (P= 0.027).
4. The combination of SLIT2,3-OST-2, CDH13 and TES provided 97.5% diagnostic coverage, which improved the sensitivity of the gene panel for cancer detection.
Conclusions:Ovarian carcinoma harbored CpG island hypermethylation of SLIT2,3-OST-2, CDH13 and TES genes. Promoter hypermethylation showed tumor-specific features and was a relatively early event in ovarian tumorigenesis. The combination of multiple genes could improve the sensitivity of the gene panel for cancer detection.
Part 2:Detecting Methylated SLIT2,3-OST-2, CDH13 and TES in Plasma for Early Diagnosis of Ovarian Carcinoma
Purpose:To determine the feasibility of circulating DNA for early diagnosis of ovarian carcinoma.
Materials and Methods:Using Methylation-specific PCR, we investigated the methylation status of SLIT2,3-OST-2, CDH13 and TES in plasma DNA from 42 patients with a probable ovarian carcinoma diagnosis, and compared the methylation status of DNA from tissues to that from plasma.
Results:
1. In the 42 patients with suspected ovarian carcinoma, the frequency of promoter hypermethylation detected in plasma was 38.1%(16/42) for SLIT2, 28.6%(12/42) for 3-OST-2,19.0%(8/42) for CDH13 and 19.0%(8/42) for TES. Overall,54.8%(23/42) of the plasma DNA samples had at least one gene promoter hypermethylated. But no methylation of any gene was detected in the 30 plasma DNA samples from healthy age-matched women.
2. After surgery, we examined the hypermethylation status of the panel genes in the matched tumor tissues.Comparing the methylation results from the plasma samples to the tumor samples revealed that the sensitivity of the gene panel was 88.5%(23/26) in plasma and 96.2%(25/26) in tissues, and the specificity both were 100%.
3. The methylation concordance between plasma and paired tumor DNA was 95.2%(40/42).
4. If plasma DNA was substituted for tumor DNA for diaagnosis, the sensitivity, specificity, accuracy, positive prognostic value and negative prognostic value were 92.0%(23/25),100%(17/17),95.2%(40/42),100%(23/23) and 89.5%(17/19), respectively.
5. The methylation frequency of at least one gene in plasma DNA form stage I-Ⅱpatients was 70%(7/10).
6. The frequency of CDH13 hypermethylation was still associated with CA 125 level (P=0.021) in tumor DNA.
Conclusions:The aberrant promoter hypermethylation of tumor suppressor genes can be detected in the plasma of ovarian carcinoma patients. Circulating hypermethylated DNA detection is a promising strategy for early detection of ovarian carcinoma.
Part 3:Evaluation of Methylated SLIT2,3-OST-2, CDH13 and TES in Ascites for Diagnosis of Ovarian Carcinoma
Purpose:To determine the feasibility of ascites DNA for diagnosis of ovarian carcinoma.
Materials and Methods:Using Methylation-specific PCR, we analysed the methylation status of SLIT2,3-OST-2, CDH13 and TES in ascites DNA from 42 patients with a probable ovarian carcinoma diagnosis, and compared the results to that from tissues and from plasma.
Results:
1.In the 42 patients with suspected ovarian carcinoma, the frequency of promoter hypermethylation detected in ascites was 47.6%(20/42) for SLIT2, 38.1%(16/42) for 3-OST-2,21.4%(9/42) for CDH13 and 23.8%(10/42) for TES. Overall,52.4%(22/42) of the ascites DNA samples had at least one gene promoter hypermethylated.
2. The sensitivity of the gene panel for ovarian carcinoma diagnosis was 84.6% (22/26) in ascites. If ascites DNA was substituted for tumor DNA for diaagnosis, the sensitivity, specificity, accuracy, positive prognostic value, negative prognostic value were 88.0%(22/25),100%(17/17),92.9%(39/42),100%(22/22) and 85.0% (17/20), respectively.
3. In the abnormal cytological patients,19 showed aberrant promoter hypermethylation, the sensitivity and specificity was 95.0%(19/20) and 100%(22/22), respectively.
4. In the benign tumor patients, no methylation of any gene was detected in their ascites DNA, which was coincident with the results of plasma.
5. The methylation frequency of panel genes in ascites DNA from stage I-II patients was 60%(6/10).
Conclusions:The aberrant promoter hypermethylation of tumor suppressor genes can be detected in the ascites of ovarian carcinoma patients. Ascites DNA is a surrogate for methylation analysis of tumor DNA or plasma DNA.
引文
1. Shen, K., Problems in connection to treatment of ovarian tumors and fertility preservation. Zhonghua Fu Chan Ke Za Zhi,2003.38(8):p.489-92.
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3. Dallol, A., et al., SLIT2, a human homologue of the Drosophila Slit2 gene, has tumor suppressor activity and is frequently inactivated in lung and breast cancers. Cancer Res,2002.62(20):p.5874-80.
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