| 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-bear101 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 chemotherapy101 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.
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