The Study Of Cell-free DNA In Ovarian Cancer Patient

Objective: Epithelial ovarian cancer (EOC) patients was the leading cause of death in the field of gynecologic malignancies .The five-year survival rate for ovarian carcinoma was about 30% all along time. Ovarian cancer is difficult to diagnose at an early stage for its delitescencal beginning. Thus, more than 75% ovarian cancer patients are at an advanced stage when discovered. The five-year survival rate for ovarian carcinoma was 85-90% for early stage ,but most patients present with advanced desease for which the long-term survival remains about 30%.At present, The most extensively studied seral tumor marker used in diagnosing and screening for ovarian cancer is CA-125 in clinical,but The CA-125 can be elevated in many other benign diseases.Thus ,it is needed a more sensitive seral index for early-diagnosing and detecting patient's condition in ovarian cancer for a long time.Recently,despite of many investers dedicted to the study of early-detecting of ovarian carcinoma but up to now no acceptable index could be used in clinical.Now, The detection of circulating cell-free DNA(cfDNA) has long been explored for the diagnosis of a variety of clinical conditions,cfDNA exist in the body fluid e.g serum or plasma and urine and released from cellular apoptosis and necrosis.But little attention was drawn to these findings until late 1980s again. Several recent studies have shown that it is possible to detect tumor-specific genetic or epigenetic alterations in serum DNA from head and neck, lung, and colon cancer patients [1-3].Importantly, tumor cell-specific DNA alterations in serum were not limited to patients with metastatic cancer but were also present in serum from patients with early or organ-confined tumors [1-3]. We used semi-quantitive PCR to study the correlation of the level of the tumor-derived cfDNA (ALU247/ALU115) and CA125-cfDNA( CA125-cfDNA,the gene of CA-125 amperlified from cfDNA fragment) which in serum of patient with epithelial ovarian carcinoma and the desease staging ,progression and the clinical pathology .Additionaly , through the study of the relation of the CA125-cfDNA and CA125 in ovarian cancer patient's serum,as a result ,we want to afford a new field for diagnosis and detecting the progression of ovarian carcinoma.Methods: Ovarian cancer patients in hospital, diagnosed by clinic and histopathology and staged by International Federation of Gynecology and Obstetrics (FIGO) Staging Systems 2000, were derived from the gynecology deparment of the Fourth Affiliated Hospital of Hebei Medical University , Dec.2005 to Mar. 2007.70 patients: being 48 with epithelial ovarian carcinoma(16 with endometrioid, 26 with serous , 6 with mucinous ),being 16 with benign epithelial ovarian tumor (11with serous ,5 with mucinous ), 12 healthy volunteer was both the paitients who subjected to uterine prolapse of the same hospital in the same times and the doctors of obsterics Dept.in the same hospital. The ovarian cancer serum samples involved preoperation postoperation and 23 after Chem of the EOC patient. Five milliter of veners blood from each subject who was on empty stomach in the early morning was stored at 4℃less than 6 hours before the certain disposal for obtained serum. The 26 ascites samples was obtained from the EOC patients (not received Chem before operation )when it was operation.Apply ANOVA (analysis of variance) ,t'test and spearman correlation of SPSS10.0 statistical software handling data. The circulating cell-free DNA fragments extracted from serum used QIAamp DNA Blood Mini Kit, followed by semi-PCR to amperlified ALU DNA repeats, CA125 DNA andβ-actin . Serum DNA integrity was assessed by polymerase chain reaction of the ratio of ALU 247/ALU115 DNA repeats,The level of cfCA-125 used cfCA-125/β-actin.Results1 The integrity of cfDNA measured as the ratio of ALU247/ALU115.The mean value of serum cfDNA relertive integrity in preoperative (0.597±0.280)or postoperative EOC was(0.617±0.158) , which was significantly higher (p<0.05) than not only that of the patients with benign tumor (0.283±0.062) and the healthy controls (0.158±0.021) but also that of the EOC patients who subjected to operation and after two-cycles chemotherapy(0.384±0.196) .The comparison of these two groups preoperative and postoperative showed no significant difference (P> 0.05),benign tumors and normal healthy control group had no significant difference .the cfDNA integrity of stage III-IV was significantly higher than stage I-II, The integrity of circulating cell-free DNA in the serum of EOC was correlate with its clinical stage (P= 0.016) but not with the pathological type and histological grade.2 The mean value of serum CA125-cfDNA relertive integrity in preoperative was(1.249±0.780) or postoperative EOC was(1.229±0.555) , which was significantly higher (p<0.05) than not only that of the patients with benign tumor (0.751±0.203) and the healthy controls (0.192±0.060) but also that of the EOC patients who subjected to operation and after chemotherapy (0.770±0.225).The comparison of these two groups preoperative and postoperative showed no significant difference (P> 0.05),benign tumors and normal healthy control group had no significant difference . the CA125-cfDNA level of stage III-IV was significantly higher than stage I-II, The level of CA125-cfDNA in the serum of EOC was correlate with its clinical stage (P<0.05) but not with the pathological type and histological grade.3 Both circulating cell-free CA125 DNA level and CA-125 of patients with EOC were correlate preoperation(r=0.878,P<0.05) but not after Chem(r=0.6,P>0.05).4 The relertive level of cell-free DNA in ascites of the advanced EOC patient was (2.84515±0.318)and about higher 2.5 times than this in serum.Conclusions1 The level of tumor-derived cfDNA and cfCA-125 of patients with ovarian cancer before operation was significantly higher than that of ovarian benign tumor patients and healthy controls, that can potentially be used to indicate their differential diagnosis of benign and malignant ovarian tumor;2 The level of tumor-derived cfDNA and cfCA-125 of patients with ovarian cancer preoperation which was significantly higher (p<0.05) than not only that of the patients after two-cycles chemothearapy and the healthy controls . The level of tumor-derived cfDNA and cfCA-125 of after two-cycles chemothearapy was significantly higher than that of the healthy controls , as a result,it is suggested that the above two markers may have correlation with the tumor sizes and could be detecting ovarian cancer.3 The value of tumor-derived cfDNA and cfCA-125 of patients with ovarian cancer of stage III-IV was significantly higher than stage I-II and correlate with its clinical stage but not with the pathological type and histological grade,the result proposed that two index may be an early-diagnosing and detecting markers.4 CA125-cfDNA and CA-125 has the same trend preoperation and after two-times chemotherapy through the correlation comparison indicate that the two group has a positive correlation. but the CA-125 expression in mucinous ovarian cancer tended to normal However CA125-cfDNA in different clinical pathologic type has no statistically significant difference, the value of after two-times chemotherapy was significant ly higher than that of the healthy controls So CA125-cfDNA could be as a supplemenin of CA125 in ovarian cancer monitoring .