循环无细胞DNA对卵巢上皮性癌患者的诊断、疗效评价和病情监测的价值
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摘要
目的:卵巢上皮性癌(epithelial ovarian cancer,EOC)早期诊断困难,5年生存率长期徘徊在30%左右。目前临床常用的诊断和监测指标是血清CA125,但由于CA125存在一定的局限性且缺乏特异性,因此长期以来临床上亟需一个更灵敏的卵巢上皮性癌诊断和病情监测的血清学指标。循环无细胞DNA(circulating cell-free DNA, cfDNA)是存在于体液中的,来源于凋亡和坏死的肿瘤细胞的碎裂核酸片段。近来的研究表明在头颈癌、肺癌、和结肠癌患者的血清中可以检测到肿瘤特异性改变的cfDNA。重要的是,这种改变不仅存在于癌症转移患者的血清中而且也存在于早期原发癌症患者的血清中。对乳腺癌的研究发现血清中的肿瘤相关cfDNA可以用ALU247/ALU115的比值代表,并证明其与疾病进展和瘤负荷呈正相关。ALU是一种重复序列,它是人类基因组中的散布元件,占基因总量的10%以上,典型的ALU重复序列有三百个碱基,能够特异性地被ALU内切酶所切割形成核酸片段,因此,理论上说,含有这一重复序列的DNA片段在血清中出现的几率最高,具有普遍意义。研究发现,循环中高表达的肿瘤相关抗原,其相应的基因组特定部位也处于高表达状态,由于肿瘤细胞的坏死和凋亡,可以将高表达的基因片段释放入循环,由此检测相应的cfDNA的数量和性质,可以为恶性肿瘤的诊断及鉴别诊断提供依据。为了找到卵巢上皮性癌患者血清中特异性的cfDNA,我们针对临床常用的诊断和监测指标血清CA125蛋白的基因序列设计了101 bp、261 bp、189 bp和336 bp四组DNA片段引物,采用半定量PCR方法检测卵巢上皮性癌患者、人卵巢癌裸鼠移植瘤模型血清和腹水中CA125基因片段(CA125-cfDNA)的含量及肿瘤相关cfDNA(ALU247/ALU115)的值,探讨其与卵巢上皮性癌鉴别诊断、临床分期,病理学类型和疾病进展的关系,研究血清中CA125-cfDNA及肿瘤相关cfDNA水平在卵巢上皮性癌临床诊断及病情监测中的意义。同时检测这两种血清cfDNA在卵巢癌、宫颈癌、子宫内膜癌、子宫肌瘤、子宫内膜异位症、复发卵巢癌、直肠癌患者血清中的含量,并与血清CA125蛋白的值进行相关性分析,横向对比分析CA125-cfDNA及肿瘤相关cfDNA是否可以辅助CA125蛋白为卵巢上皮性癌的诊断、疗效评价和病情监测提供依据。
本实验共分为三个部分:
第一部分卵巢上皮性癌患者循环无细胞DNA的检测及临床意义目的:检测循环无细胞DNA中的肿瘤相关cfDNA(ALU247/ALU115)及CA-125基因片段(CA125-cfDNA)在卵巢上皮性癌患者血清中的含量,探讨其与卵巢上皮性癌鉴别诊断、临床分期,病理学类型和疾病进展的关系,研究二者是否可作为卵巢上皮性癌早期诊断及病情监测的指标。
方法:采用半定量PCR方法检测60例卵巢上皮性癌,35例良性卵巢上皮性肿瘤,30例正常对照组血清及26例晚期卵巢癌患者未经化疗腹水中游离的ALU 247、ALU115、β-actin、CA125 DNA片段的表达。血清CA125的检测为常规的酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA)检测法。
结果:1肿瘤相关cfDNA、CA125-cfDNA在卵巢上皮性癌组、卵巢良性肿瘤组和正常对照组血清中的表达
卵巢上皮性癌患者术前血清肿瘤相关cfDNA、CA125-cfDNA的水平显著高于正常对照组(P=0.000, P<0.05),CA125-cfDNA在卵巢良性肿瘤的含量与正常对照组相比无统计学差异(P=1.000, P>0.05),肿瘤相关cfDNA在卵巢良性肿瘤的表达高于正常对照组(P=0.019, P<0.05)。血清CA125-cfDNA在晚期卵巢上皮性癌患者腹水中的值显著高于同组血清中的值(P=1.000, P<0.05)。
2肿瘤相关cfDNA、CA125-cfDNA在卵巢上皮性癌患者术前、术后及化疗两程后血清中的含量卵巢上皮性癌患者血清术前及术后肿瘤相关cfDNA、CA125-cfDNA的水平显著高于正常对照组及术后化疗二程组(P=0.009, P<0.05),化疗二程后明显高于健康对照组(P=0.012, P<0.05),术前、术后比较无显著性差异(P=0.953, P>0.05)。
3肿瘤相关cfDNA、CA125-cfDNA含量与卵巢上皮性癌手术病理分期、病理学分级和类型的关系
III-IV期卵巢上皮性癌患者的肿瘤相关cfDNA和CA125-cfDNA的水平显著高于I-II期(t=-4.250, P=0,000, P<0.05),二者与卵巢上皮性癌的病理学类型及病理分级间无统计学差异(F=2.641, P=0.08, P>0.05)。血清CA125-cfDNA在卵巢粘液性囊腺癌中的水平显著高于正常对照组(t=4.605, P=0.001, P<0.05)。
4卵巢癌肿瘤相关cfDNA、CA125-cfDNA水平与血清CA125的相关性比较
卵巢上皮性癌患者术前血清CA125的值与血清CA125-cfDNA的值存在相关性(P<0.05),与肿瘤相关cfDNA的值无相关性(P>0.05)。卵巢上皮性癌患者化疗2疗程后血清CA125的值与血清CA125-cfDNA的值存在相关性(P<0.05),与肿瘤相关cfDNA的值无相关性(P>0.05)。
结论:1肿瘤相关cfDNA、CA125-cfDNA在卵巢上皮性癌患者血清中水平显著升高,可能与瘤负荷呈正相关。2血清CA125-cfDNA术前和化疗两疗程后的值与血清CA125的值有较好的相关性,而肿瘤相关cfDNA与血清CA125不存在相关性,因此肿瘤相关cfDNA和CA125-cfDNA有望成为卵巢上皮性癌病情监测新的分子标记物,可以辅助血清CA125用于卵巢上皮性癌的诊断、鉴别诊断和病情监测。3 CA125在粘液性卵巢癌中的表达往往趋于正常值,然而在CA125的值正常的4例粘液性卵巢癌中,CA125-cfDNA有2例较正常均值有明显升高,肿瘤相关cfDNA有3例升高,因此二者可能在卵巢粘液性癌的诊断及病情监测中发挥重要作用。
第二部分人卵巢癌移植瘤裸鼠模型中循环无细胞DNA与瘤负荷、凋亡的关系及其血流动力学改变目的:研究人卵巢癌移植瘤裸鼠模型血清及腹水中cfDNA的血流动力学改变及其与瘤负荷和癌细胞凋亡的关系。
方法:将人卵巢癌细胞株SKOV3种植于6-8周龄雌性裸鼠的皮下及腹腔中,观察裸鼠成瘤和肿瘤生长情况。分别于14天(荷瘤1组),21天(荷瘤2组)和30天(荷瘤3组)取皮下种植瘤模型血清及肿瘤,14天取腹水种植瘤模型血清和腹水,皮下种植瘤模型化疗组在种植后第30天用顺铂化疗1次,于化疗后第1天(化疗1组),第3天(化疗2组)和第10天(化疗3组)取血清及肿瘤。应用半定量PCR法检测荷瘤1、2、3组;化疗1、2、3组和腹水组的血清及腹水组腹水中cfDNA的含量,与相应的肿瘤重量进行对比,用TUNNEL法检测化疗1、2、3组及荷瘤
1组组织细胞的原位凋亡,将其凋亡率与相应的cfDNA含量及重量进行相关性分析。
结果:
1各组基因片段在成瘤裸鼠血清及腹水中的表达情况裸鼠血清及腹水中β-actin、ALU247、ALU115、以及CA125-cfDNA中的101bp片段在各组均有表达,CA125-cfDNA中的261bp片段表达阳性率达51.25%(80例中有41例可见特异性条带),189 bp及336 bp片段基本没有表达。CA125-cfDNA101 bp及肿瘤相关cfDNA在腹水中的表达与同一个体的血清值相比没有显著性差异(P>0.05)。
2血清cfDNA水平与瘤负荷的关系瘤负荷1、2、3组裸鼠血清中的CA125-cfDNA 101bp片段及肿瘤相关cfDNA与肿瘤重量呈正相关(CA125-cfDNA 101bp片段R=0.830,肿瘤相关cfDNA的R=780),差异均有显著性(p<0.05)。
3血清cfDNA在化疗后的变化情况
血清CA125-cfDNA的101 bp片段及肿瘤相关cfDNA在化疗后第1天达最大值,而后缓慢下降,在化疗第10天其值明显低于化疗前水平,差异有显著性(CA125-cfDNA的P=0.047,肿瘤相关cfDNA的P=0.013,均<0.05)。
4化疗后血清cfDNA水平与肿瘤细胞凋亡的关系
化疗前后血清CA125-cfDNA 101 bp片段及肿瘤相关cfDNA与肿瘤细胞的凋亡率呈正相关(P<0.05)。
结论: 1成瘤裸鼠血清及腹水中可以检测到人卵巢癌细胞株的人类特异性cfDNA,其含量在化疗后先显著升高而后下降,并与tunel检测的凋亡率呈正相关,由此可以证实cfDNA来源于肿瘤细胞,并与其凋亡和坏死有关。2血清中CA125-cfDNA中101 bp片段及肿瘤相关cfDNA的含量与瘤负荷呈正相关,有可能为卵巢上皮性癌的诊断及病情监测提供理论依据。
第三部分循环无细胞DNA与CA125的相关性研究及其在妇科各类疾病中的表达状况分析
目的:对比妇科各类良恶性疾病及结直肠癌患者血清的肿瘤相关cfDNA及CA125-cfDNA的含量,并与血清CA125的值进行相关性分析。
方法:应用半定量PCR法检测60例卵巢癌、18例宫颈癌、15例子宫内膜癌、15例子宫肌瘤、26例子宫内膜异位症、8例复发卵巢癌、12例结直肠癌患者、35例卵巢良性肿瘤患者及30例正常人血清中肿瘤相关cfDNA及CA125-cfDNA261 bp片段的含量,分析这两项指标在妇科各类良恶性疾病中的表达情况及与血清CA125蛋白含量的相关性,进而推断二者是否可以辅助血清CA125成为卵巢上皮性癌的特异性检测指标。
结果:
1血清CA125-cfDNA在各类疾病中的含量变化及与CA125的相关性分析血清CA125-cfDNA的含量在卵巢癌、复发性卵巢癌中显著升高,与正常人及卵巢良性肿瘤有显著性差异(P<0.05)。在子宫内膜癌、子宫内膜异位症及结直肠癌中表达轻度升高,与正常人相比有显著性差异(P<0.05)。在宫颈癌、子宫肌瘤的患者血清中表达极少。血清CA125-cfDNA的含量与血清CA125蛋白存在正相关关系(P<0.05)。
2血清肿瘤相关cfDNA在各类疾病中的含量变化及与CA125的相关性分析
血清肿瘤相关cfDNA的含量在卵巢癌、复发性卵巢癌、直肠癌、子宫内膜癌及宫颈癌等各类实体瘤性癌症患者血清中均有升高,各组癌症患者之间比较,差异没有显著性(P>0.05)。各组与卵巢良性肿瘤组和正常对照组相比,差异有显著性(P<0.05)。与CA125不存在正相关关系(P>0.05)。
结论:1血清CA125-cfDNA及肿瘤相关cfDNA的检测可以辅助CA125在卵巢癌患者的诊断及病情监测中具有临床应用价值。2血清肿瘤相关cfDNA是否可以作为一项癌症的普查手段尚需进一步研究证实。
90% ovarian malignant tumor is epithelial ovarian carcinoma (EOC), which is the first most common cause of female genital cancer related death, and the five-year survival is 30%. The most commonly used biomarker for diagnosis and assessment the therapeutic response is serum CA125. But CA125 is not entirely satisfactory for early detection and diagnosis for EOC patients because of the lack of sufficient specificity and sensitivity. It is possible that circulating cell-free DNA (cfDNA) has the clinical potential to be a more specific tumor biomarker for the diagnosis and prognosis, as well as the early detection, of patient with EOC.Circulating cfDNA is detectable DNA sequence fragment in plasma or serum of healthy individuals or patients, probably through cellular apoptosis and tumor cellular necrosis. In the next 5 years, several reports related to the detection of high fractional concentrations of tumor DNA in the plasma (or serum) of patients with various human cancers. ALU repeated DNA sequence. ALU repeats are the most abundant sequences in the human genome, with a copy number of about 1.4 million per genome. ALU sequences are short interspersed elements (SINEs), typically 300 nucleotides, which account for more than 10% of the genome. In one article in 2006, DNA integrity was calculated as a ratio of longer to shorter DNA fragments(ALU247/ALU115), which was called tumor related cell-free DNA. Mean serum DNA integrity was significantly higher in patients with LVI-positive tumors and had a highly significant predictive value for LN metastasis in breast cancer in that article. Increased DNA copy number or genomic amplification at certain loci are frequently present in a variety of human cancers. As genomic amplification occurs only in cancer but not in normal tissues, measurement of a certain amplified chromosomal region may likely increase both the sensitivity and specificity of the DNA copy number and DNA strand integrityassays. In the current report, CA125 cell-free DNA( CA125-cfDNA), in the serum of patients with epithelial ovarian carcinoma (EOC) ,was first detected by our group. We designed four primers base on the CA-125 gene sequence(261 bp,189 bp,336 bp and 101 bp )and in our result, the 252 bp fragment showed better specificity than the other three fragments. Semi-quantitive PCR was used to analyze the level of the serum cfDNA, including ALU247, ALU115, CA125-cfDNA andβ-actin. 48 patients with EOC ( 16 endometrioid, 26 serous and 6 mucinous adenocarcinomas), 16 patients with benign ovarian tumor(11 serous and 5 mucinous cystadenomas)and 12 normal controls were involved. All of EOC patients were diagnosed by histopathologist and were staged by FIGO2000. We used QIAamp DNA Blood Mini Kit to extract serum cfDNA. During serum separation, cell lysis due to processing may release long DNA fragments into the serum and raise the DNA integrity.
Part 1. The clinical significance of detecting and exploring serum Cell-free DNA in patients with epithelial ovarian carcinoma.
Objective:To detect the tumor related cell-free DNA (ALU247/ALU115)and a gene fragment of CA125, CA125 cell-free DNA( CA125-cfDNA) in the serum of patients with epithelial ovarian carcinoma (EOC) and to investigate the values to early diagnosis and prognosis in EOC.
Methods:Semi-quantitive PCR was used to analyze the level of the serum cell-free DNA(cfDNA), including ALU247, ALU115, CA125-cfDNA andβ-actin in 48 patients with epithelial ovarian carcinoma (EOC), 16 patients with benign ovarian tumor and 12 normal controls.
Results : 1 The mean values of serum tumor related cfDNA and CA125-cfDNA in the pre-operative patients with EOC were significantly higher than those of the patients with benign tumor, the control group and the patients after operation and two-cycle chemotherapy. The mean values of serum tumor related cell-free DNA and CA125-cfDNA in EOC patients of stage III-IV were significantly higher than those of stage I-II (p<0.05). There were no correlations of tumor related cell-free DNA and CA125-cfDNA levels in the serum with clinical stage ,the pathological type and histological grad(eP>0.05). The mean value of CA125-cfDNA in serum was correlated with serum CA125, and it was much higher in ascites of patients with advanced EOC than that in the serum (P<0.05).
Conclusion:1. The level of tumor related cfDNA and CA125-cfDNA were increased in serum,and it may be positive related with tumor bear. 2. Preoperative or after two period chemotherapy serum CA125 level correlated to CA125-cfDNA level very well, but not correlated to tumor related cfDNA.
The serum tumor related cfDNA (ALU247/ALU115)and CA125-cfDNA may be used as new markers associated serum CA125 in early diagnosis and prognosis of patients with EOC. 3. Of 4 mucinus carcinomas with normal level of serum CA125, 2 were found with high CA125-cfDNA level, and 3 were found with tumor related cfDNA. The serum CA125 level in mucinus carcinoma close to normal level commonly, so CA125-cfDNA and tumor related cfDNA may play important role in diagnosis and prognosis of mucinus carcinoma. 4. CA125-cfDNA level in ascites of advanced stage EOC is significantly higher than in the other non-cancer ascites, it may be use for differentiation of benign and malignant ascites as a aid means.
Part 2. Investigate whether tumor related cfDNA correlate to tumor bear or apoptosis and its’blood flow kinetics in animal model.
Objective:Investigate the change of cfDNA blood flow kinetics in the serum of female nude mice after human EOC cell line injection. Methods 6-8 weeks female nude mice were injected on the back and intra-abdomen by human EOC cell line SKOV3, the tumors and serum were collected on day 14, day 21 and day 30 respectively in tumor-bear group; The serum and ascites were collected on day 30 in ascites group; the mice were given cisplatin injection on day 30, the serum and tumors were collected on day 22, day 25, day 31 in chemotherapy group. Semi-quantitive PCR was used to analysis cfDNA level in the serum, TUNNEL was used to detect cell in-situ apoptosis. The correlation between cfDNA level in the serum or ascites and tumor volume or apoptosis were analysis.
Results: 1.DNA fragments of ALU247, ALU115, 101bp fragment of CA125-cfDNA andβ-actin were found in the serum or ascites of every groups, especially the 261 bps fragment of CA125-cfDNA level was found in 41 of 80 mice (51.25%), but the 189 bps and 336 bps fragments were not found in all groups.
2. Serum cfDNA level and tumor-bear
101 bps fragment of CA125-cfDNA and tumor related cfDNA in serum are positive correlated with tumor weight, the differences are significant in statistics(P<0.05).
3. Serum cfDNA level detection after chemotherapy
101 bps fragment of CA125-cfDNA and tumor related cfDNA in serum reach to maximal value on day 1 after chemotherapy, and then slow down, the values were significant lower on day 10 after chemotherapy than that pre-chemotherapy. 4. Serum cfDNA level and tumor cell apoptosis after chemotherapy. 101 bps fragment of CA125-cfDNA and tumor related cfDNA are positive correlated with cell apoptosis respectively. (P<0.05)
Conclusion:1. Human specific cfDNA can be found in the serum and ascites of tumor-bear nude mice, the cfDNA level increased significantly after chemotherapy and then slow down, also it correlated with cell apoptosis. Base on our results, the cfDNA coming from tumor cell was certified, and it also correlated with apoptosis and necrosis. 2. 101 bps fragment of CA125-cfDNA and tumor related cfDNA in serum positive correlated with tumor-bear, showed some useful information to EOC diagnosis and management.
Part 3. Analysis of correlation between cell-free DNA and CA125 level, and its’level in the other gynecological diseases.
Objective: Investigate the level of cell-free DNA and CA125 in the serum of various gynecological diseases.
Methods: We detected the level of cell-free DNA and CA125 in the serum of 60 ovarian cancers, 18 cervix cancers, 15 endometrium cancers, 15 leiomyomas, 26 endometriosis, 8 recurrent ovarian cancers and 12 colorectal cancers.
Results:CA125-cfDNA level in serum was significant increased in ovarian cancer, recurrent ovarian cancers and rectum cancer, slightly increased in endometrium cancer and endometriosis, almost non appear in cervix cancer and leiomyoma, and it’s level correlated with CA-125 level. (P<0.05)2. Tumor related cfDNA in serum is increased in solid tumor like ovarian cancer, recurrent ovarian cancers, endometrium cance, cervix cancer and rectum cancer, but the level is not correlated with CA-125 level and lack specificity. (P>0.05)Ther is no significant difference between various cancer patients. (P>0.05)
Conclusion:1. Detection of CA125-cfDNA and tumor related cfDNA level in serum, combine CA-125 level, provide some clinical applications. 2. More work need to be done to decide whether tumor related cfDNA level can be use for cancer population exam.
本实验共分为三个部分:
第一部分卵巢上皮性癌患者循环无细胞DNA的检测及临床意义目的:检测循环无细胞DNA中的肿瘤相关cfDNA(ALU247/ALU115)及CA-125基因片段(CA125-cfDNA)在卵巢上皮性癌患者血清中的含量,探讨其与卵巢上皮性癌鉴别诊断、临床分期,病理学类型和疾病进展的关系,研究二者是否可作为卵巢上皮性癌早期诊断及病情监测的指标。
方法:采用半定量PCR方法检测60例卵巢上皮性癌,35例良性卵巢上皮性肿瘤,30例正常对照组血清及26例晚期卵巢癌患者未经化疗腹水中游离的ALU 247、ALU115、β-actin、CA125 DNA片段的表达。血清CA125的检测为常规的酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA)检测法。
结果:1肿瘤相关cfDNA、CA125-cfDNA在卵巢上皮性癌组、卵巢良性肿瘤组和正常对照组血清中的表达
卵巢上皮性癌患者术前血清肿瘤相关cfDNA、CA125-cfDNA的水平显著高于正常对照组(P=0.000, P<0.05),CA125-cfDNA在卵巢良性肿瘤的含量与正常对照组相比无统计学差异(P=1.000, P>0.05),肿瘤相关cfDNA在卵巢良性肿瘤的表达高于正常对照组(P=0.019, P<0.05)。血清CA125-cfDNA在晚期卵巢上皮性癌患者腹水中的值显著高于同组血清中的值(P=1.000, P<0.05)。
2肿瘤相关cfDNA、CA125-cfDNA在卵巢上皮性癌患者术前、术后及化疗两程后血清中的含量卵巢上皮性癌患者血清术前及术后肿瘤相关cfDNA、CA125-cfDNA的水平显著高于正常对照组及术后化疗二程组(P=0.009, P<0.05),化疗二程后明显高于健康对照组(P=0.012, P<0.05),术前、术后比较无显著性差异(P=0.953, P>0.05)。
3肿瘤相关cfDNA、CA125-cfDNA含量与卵巢上皮性癌手术病理分期、病理学分级和类型的关系
III-IV期卵巢上皮性癌患者的肿瘤相关cfDNA和CA125-cfDNA的水平显著高于I-II期(t=-4.250, P=0,000, P<0.05),二者与卵巢上皮性癌的病理学类型及病理分级间无统计学差异(F=2.641, P=0.08, P>0.05)。血清CA125-cfDNA在卵巢粘液性囊腺癌中的水平显著高于正常对照组(t=4.605, P=0.001, P<0.05)。
4卵巢癌肿瘤相关cfDNA、CA125-cfDNA水平与血清CA125的相关性比较
卵巢上皮性癌患者术前血清CA125的值与血清CA125-cfDNA的值存在相关性(P<0.05),与肿瘤相关cfDNA的值无相关性(P>0.05)。卵巢上皮性癌患者化疗2疗程后血清CA125的值与血清CA125-cfDNA的值存在相关性(P<0.05),与肿瘤相关cfDNA的值无相关性(P>0.05)。
结论:1肿瘤相关cfDNA、CA125-cfDNA在卵巢上皮性癌患者血清中水平显著升高,可能与瘤负荷呈正相关。2血清CA125-cfDNA术前和化疗两疗程后的值与血清CA125的值有较好的相关性,而肿瘤相关cfDNA与血清CA125不存在相关性,因此肿瘤相关cfDNA和CA125-cfDNA有望成为卵巢上皮性癌病情监测新的分子标记物,可以辅助血清CA125用于卵巢上皮性癌的诊断、鉴别诊断和病情监测。3 CA125在粘液性卵巢癌中的表达往往趋于正常值,然而在CA125的值正常的4例粘液性卵巢癌中,CA125-cfDNA有2例较正常均值有明显升高,肿瘤相关cfDNA有3例升高,因此二者可能在卵巢粘液性癌的诊断及病情监测中发挥重要作用。
第二部分人卵巢癌移植瘤裸鼠模型中循环无细胞DNA与瘤负荷、凋亡的关系及其血流动力学改变目的:研究人卵巢癌移植瘤裸鼠模型血清及腹水中cfDNA的血流动力学改变及其与瘤负荷和癌细胞凋亡的关系。
方法:将人卵巢癌细胞株SKOV3种植于6-8周龄雌性裸鼠的皮下及腹腔中,观察裸鼠成瘤和肿瘤生长情况。分别于14天(荷瘤1组),21天(荷瘤2组)和30天(荷瘤3组)取皮下种植瘤模型血清及肿瘤,14天取腹水种植瘤模型血清和腹水,皮下种植瘤模型化疗组在种植后第30天用顺铂化疗1次,于化疗后第1天(化疗1组),第3天(化疗2组)和第10天(化疗3组)取血清及肿瘤。应用半定量PCR法检测荷瘤1、2、3组;化疗1、2、3组和腹水组的血清及腹水组腹水中cfDNA的含量,与相应的肿瘤重量进行对比,用TUNNEL法检测化疗1、2、3组及荷瘤
1组组织细胞的原位凋亡,将其凋亡率与相应的cfDNA含量及重量进行相关性分析。
结果:
1各组基因片段在成瘤裸鼠血清及腹水中的表达情况裸鼠血清及腹水中β-actin、ALU247、ALU115、以及CA125-cfDNA中的101bp片段在各组均有表达,CA125-cfDNA中的261bp片段表达阳性率达51.25%(80例中有41例可见特异性条带),189 bp及336 bp片段基本没有表达。CA125-cfDNA101 bp及肿瘤相关cfDNA在腹水中的表达与同一个体的血清值相比没有显著性差异(P>0.05)。
2血清cfDNA水平与瘤负荷的关系瘤负荷1、2、3组裸鼠血清中的CA125-cfDNA 101bp片段及肿瘤相关cfDNA与肿瘤重量呈正相关(CA125-cfDNA 101bp片段R=0.830,肿瘤相关cfDNA的R=780),差异均有显著性(p<0.05)。
3血清cfDNA在化疗后的变化情况
血清CA125-cfDNA的101 bp片段及肿瘤相关cfDNA在化疗后第1天达最大值,而后缓慢下降,在化疗第10天其值明显低于化疗前水平,差异有显著性(CA125-cfDNA的P=0.047,肿瘤相关cfDNA的P=0.013,均<0.05)。
4化疗后血清cfDNA水平与肿瘤细胞凋亡的关系
化疗前后血清CA125-cfDNA 101 bp片段及肿瘤相关cfDNA与肿瘤细胞的凋亡率呈正相关(P<0.05)。
结论: 1成瘤裸鼠血清及腹水中可以检测到人卵巢癌细胞株的人类特异性cfDNA,其含量在化疗后先显著升高而后下降,并与tunel检测的凋亡率呈正相关,由此可以证实cfDNA来源于肿瘤细胞,并与其凋亡和坏死有关。2血清中CA125-cfDNA中101 bp片段及肿瘤相关cfDNA的含量与瘤负荷呈正相关,有可能为卵巢上皮性癌的诊断及病情监测提供理论依据。
第三部分循环无细胞DNA与CA125的相关性研究及其在妇科各类疾病中的表达状况分析
目的:对比妇科各类良恶性疾病及结直肠癌患者血清的肿瘤相关cfDNA及CA125-cfDNA的含量,并与血清CA125的值进行相关性分析。
方法:应用半定量PCR法检测60例卵巢癌、18例宫颈癌、15例子宫内膜癌、15例子宫肌瘤、26例子宫内膜异位症、8例复发卵巢癌、12例结直肠癌患者、35例卵巢良性肿瘤患者及30例正常人血清中肿瘤相关cfDNA及CA125-cfDNA261 bp片段的含量,分析这两项指标在妇科各类良恶性疾病中的表达情况及与血清CA125蛋白含量的相关性,进而推断二者是否可以辅助血清CA125成为卵巢上皮性癌的特异性检测指标。
结果:
1血清CA125-cfDNA在各类疾病中的含量变化及与CA125的相关性分析血清CA125-cfDNA的含量在卵巢癌、复发性卵巢癌中显著升高,与正常人及卵巢良性肿瘤有显著性差异(P<0.05)。在子宫内膜癌、子宫内膜异位症及结直肠癌中表达轻度升高,与正常人相比有显著性差异(P<0.05)。在宫颈癌、子宫肌瘤的患者血清中表达极少。血清CA125-cfDNA的含量与血清CA125蛋白存在正相关关系(P<0.05)。
2血清肿瘤相关cfDNA在各类疾病中的含量变化及与CA125的相关性分析
血清肿瘤相关cfDNA的含量在卵巢癌、复发性卵巢癌、直肠癌、子宫内膜癌及宫颈癌等各类实体瘤性癌症患者血清中均有升高,各组癌症患者之间比较,差异没有显著性(P>0.05)。各组与卵巢良性肿瘤组和正常对照组相比,差异有显著性(P<0.05)。与CA125不存在正相关关系(P>0.05)。
结论:1血清CA125-cfDNA及肿瘤相关cfDNA的检测可以辅助CA125在卵巢癌患者的诊断及病情监测中具有临床应用价值。2血清肿瘤相关cfDNA是否可以作为一项癌症的普查手段尚需进一步研究证实。
90% ovarian malignant tumor is epithelial ovarian carcinoma (EOC), which is the first most common cause of female genital cancer related death, and the five-year survival is 30%. The most commonly used biomarker for diagnosis and assessment the therapeutic response is serum CA125. But CA125 is not entirely satisfactory for early detection and diagnosis for EOC patients because of the lack of sufficient specificity and sensitivity. It is possible that circulating cell-free DNA (cfDNA) has the clinical potential to be a more specific tumor biomarker for the diagnosis and prognosis, as well as the early detection, of patient with EOC.Circulating cfDNA is detectable DNA sequence fragment in plasma or serum of healthy individuals or patients, probably through cellular apoptosis and tumor cellular necrosis. In the next 5 years, several reports related to the detection of high fractional concentrations of tumor DNA in the plasma (or serum) of patients with various human cancers. ALU repeated DNA sequence. ALU repeats are the most abundant sequences in the human genome, with a copy number of about 1.4 million per genome. ALU sequences are short interspersed elements (SINEs), typically 300 nucleotides, which account for more than 10% of the genome. In one article in 2006, DNA integrity was calculated as a ratio of longer to shorter DNA fragments(ALU247/ALU115), which was called tumor related cell-free DNA. Mean serum DNA integrity was significantly higher in patients with LVI-positive tumors and had a highly significant predictive value for LN metastasis in breast cancer in that article. Increased DNA copy number or genomic amplification at certain loci are frequently present in a variety of human cancers. As genomic amplification occurs only in cancer but not in normal tissues, measurement of a certain amplified chromosomal region may likely increase both the sensitivity and specificity of the DNA copy number and DNA strand integrityassays. In the current report, CA125 cell-free DNA( CA125-cfDNA), in the serum of patients with epithelial ovarian carcinoma (EOC) ,was first detected by our group. We designed four primers base on the CA-125 gene sequence(261 bp,189 bp,336 bp and 101 bp )and in our result, the 252 bp fragment showed better specificity than the other three fragments. Semi-quantitive PCR was used to analyze the level of the serum cfDNA, including ALU247, ALU115, CA125-cfDNA andβ-actin. 48 patients with EOC ( 16 endometrioid, 26 serous and 6 mucinous adenocarcinomas), 16 patients with benign ovarian tumor(11 serous and 5 mucinous cystadenomas)and 12 normal controls were involved. All of EOC patients were diagnosed by histopathologist and were staged by FIGO2000. We used QIAamp DNA Blood Mini Kit to extract serum cfDNA. During serum separation, cell lysis due to processing may release long DNA fragments into the serum and raise the DNA integrity.
Part 1. The clinical significance of detecting and exploring serum Cell-free DNA in patients with epithelial ovarian carcinoma.
Objective:To detect the tumor related cell-free DNA (ALU247/ALU115)and a gene fragment of CA125, CA125 cell-free DNA( CA125-cfDNA) in the serum of patients with epithelial ovarian carcinoma (EOC) and to investigate the values to early diagnosis and prognosis in EOC.
Methods:Semi-quantitive PCR was used to analyze the level of the serum cell-free DNA(cfDNA), including ALU247, ALU115, CA125-cfDNA andβ-actin in 48 patients with epithelial ovarian carcinoma (EOC), 16 patients with benign ovarian tumor and 12 normal controls.
Results : 1 The mean values of serum tumor related cfDNA and CA125-cfDNA in the pre-operative patients with EOC were significantly higher than those of the patients with benign tumor, the control group and the patients after operation and two-cycle chemotherapy. The mean values of serum tumor related cell-free DNA and CA125-cfDNA in EOC patients of stage III-IV were significantly higher than those of stage I-II (p<0.05). There were no correlations of tumor related cell-free DNA and CA125-cfDNA levels in the serum with clinical stage ,the pathological type and histological grad(eP>0.05). The mean value of CA125-cfDNA in serum was correlated with serum CA125, and it was much higher in ascites of patients with advanced EOC than that in the serum (P<0.05).
Conclusion:1. The level of tumor related cfDNA and CA125-cfDNA were increased in serum,and it may be positive related with tumor bear. 2. Preoperative or after two period chemotherapy serum CA125 level correlated to CA125-cfDNA level very well, but not correlated to tumor related cfDNA.
The serum tumor related cfDNA (ALU247/ALU115)and CA125-cfDNA may be used as new markers associated serum CA125 in early diagnosis and prognosis of patients with EOC. 3. Of 4 mucinus carcinomas with normal level of serum CA125, 2 were found with high CA125-cfDNA level, and 3 were found with tumor related cfDNA. The serum CA125 level in mucinus carcinoma close to normal level commonly, so CA125-cfDNA and tumor related cfDNA may play important role in diagnosis and prognosis of mucinus carcinoma. 4. CA125-cfDNA level in ascites of advanced stage EOC is significantly higher than in the other non-cancer ascites, it may be use for differentiation of benign and malignant ascites as a aid means.
Part 2. Investigate whether tumor related cfDNA correlate to tumor bear or apoptosis and its’blood flow kinetics in animal model.
Objective:Investigate the change of cfDNA blood flow kinetics in the serum of female nude mice after human EOC cell line injection. Methods 6-8 weeks female nude mice were injected on the back and intra-abdomen by human EOC cell line SKOV3, the tumors and serum were collected on day 14, day 21 and day 30 respectively in tumor-bear group; The serum and ascites were collected on day 30 in ascites group; the mice were given cisplatin injection on day 30, the serum and tumors were collected on day 22, day 25, day 31 in chemotherapy group. Semi-quantitive PCR was used to analysis cfDNA level in the serum, TUNNEL was used to detect cell in-situ apoptosis. The correlation between cfDNA level in the serum or ascites and tumor volume or apoptosis were analysis.
Results: 1.DNA fragments of ALU247, ALU115, 101bp fragment of CA125-cfDNA andβ-actin were found in the serum or ascites of every groups, especially the 261 bps fragment of CA125-cfDNA level was found in 41 of 80 mice (51.25%), but the 189 bps and 336 bps fragments were not found in all groups.
2. Serum cfDNA level and tumor-bear
101 bps fragment of CA125-cfDNA and tumor related cfDNA in serum are positive correlated with tumor weight, the differences are significant in statistics(P<0.05).
3. Serum cfDNA level detection after chemotherapy
101 bps fragment of CA125-cfDNA and tumor related cfDNA in serum reach to maximal value on day 1 after chemotherapy, and then slow down, the values were significant lower on day 10 after chemotherapy than that pre-chemotherapy. 4. Serum cfDNA level and tumor cell apoptosis after chemotherapy. 101 bps fragment of CA125-cfDNA and tumor related cfDNA are positive correlated with cell apoptosis respectively. (P<0.05)
Conclusion:1. Human specific cfDNA can be found in the serum and ascites of tumor-bear nude mice, the cfDNA level increased significantly after chemotherapy and then slow down, also it correlated with cell apoptosis. Base on our results, the cfDNA coming from tumor cell was certified, and it also correlated with apoptosis and necrosis. 2. 101 bps fragment of CA125-cfDNA and tumor related cfDNA in serum positive correlated with tumor-bear, showed some useful information to EOC diagnosis and management.
Part 3. Analysis of correlation between cell-free DNA and CA125 level, and its’level in the other gynecological diseases.
Objective: Investigate the level of cell-free DNA and CA125 in the serum of various gynecological diseases.
Methods: We detected the level of cell-free DNA and CA125 in the serum of 60 ovarian cancers, 18 cervix cancers, 15 endometrium cancers, 15 leiomyomas, 26 endometriosis, 8 recurrent ovarian cancers and 12 colorectal cancers.
Results:CA125-cfDNA level in serum was significant increased in ovarian cancer, recurrent ovarian cancers and rectum cancer, slightly increased in endometrium cancer and endometriosis, almost non appear in cervix cancer and leiomyoma, and it’s level correlated with CA-125 level. (P<0.05)2. Tumor related cfDNA in serum is increased in solid tumor like ovarian cancer, recurrent ovarian cancers, endometrium cance, cervix cancer and rectum cancer, but the level is not correlated with CA-125 level and lack specificity. (P>0.05)Ther is no significant difference between various cancer patients. (P>0.05)
Conclusion:1. Detection of CA125-cfDNA and tumor related cfDNA level in serum, combine CA-125 level, provide some clinical applications. 2. More work need to be done to decide whether tumor related cfDNA level can be use for cancer population exam.
引文
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1 Jahr S, HeJahrntze H, Englisch S, Hardt D, fackelmayer FO, Hesch RD, Knippers R. DNA fragments in the blood plasma of cancer patients: quantitation and evidence of their origin from apoptotic and necrotic cells. Cancer Res , 2001, 61:1659~1665
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4 Umetani N, Giuliano AE, Hiramatsu SH, et al. Prediction of breast tumor progression by integrity of free circulating DNA in serum . Clin Oncol, 2006, 24: 4270~4276
5 Goebel G, Zitt M, Muller HM. Circulating nucleic acids in plasma or serum (CNAPS) as prognostic and predictive markers in patients with solid neoplasias. Dis Markers , 2005, 21: 105~120
6 Garcia-Olmo D, Garcia-Olmo DC, Ontanon J, Martinez E, Vallejo M. Tumor DNA circulating in the plasma might play a role in metastasis. The hypothesis of the genometastasis. Histol Histopathol , 1999, 14: 1159~1164
7 Ziegler A, Zangemeister-Wittke U, Stahel RA. Circulating DNA: a new diagnostic gold mine. Cancer Treat Rev , 2002, 28: 255~271
8 Ugur Deligezer, Yesim Eralp.et al. Size distribution of circulating cell-free DNA in sera of breast cancer patients in the course of adjuvant chemotherapy. Clin Chem Lab Med, 2008, 46: 311~317
9 levels of circulating cell-free serum DNA in benign and malignant breast lesions .The International Journal of Biological Markers, 22: 95~99
10 Stroun, M. and Anker, P. Nucleic acids spontaneously released by living frog auricles. Biochem, 1972, 128: 100~101
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