The Clinical Significance Of Detecting Serum Free DNA In Ovarian Patients

Objective:Circulating tumor DNA?ctDNA?is single or double chain DNA released by the tumor cells into the blood and thus harbors the mutations of the original tumor.Recently,groundbreaking studies on ctDNA have been carried out and became a promising candidate biomarker for detection,monitoring and prognostic prediction of malignant tumours.Some Studies have shown that circulating DNA levels in serum may have a potential marker in primary colorectal cancer and esophageal cancer.Due to no good indicators for the early detection of ovarian cancer,the majority of diagnosed ovarian cancers are in advanced stage.So,this study wanted to investigate whether ctDNA concentration detection have clinical significance for diagnosis of ovarian cancer patients.Additionally,the human epididymis protein 4?HE4?in serum was also detected and the combined effect with ctDNA for diagnosis of ovarian cancer was observed.Methods:The experimental design was carried out using case and control method.Serum samples were collected from 119 with primary ovarian cancer in this experiments,119 confirmed ovarian cancer?24 cases from the second hospital of Hebei Medical University,85 cases from the forth hospital of Hebei Medical University,10 cases from Hebei general hospital?and 103 healthy control?53 cases from Hebei general hospital,50 cases from the second hospital of Hebei Medical University?were accepted and the serum samples were collected and cryopreserved for-80?.Firstly,normal human genomic DNA was made 10 times serial dilution(10⁴¹0^-3 ng/mL)and was used as templates for qPCR with Alu115 and Alu247 primers,respectively.Then,the Log value of the DNA concentration was used as the horizontal ordinate,the Ct value was used as the vertical coordinates to draw the standard curve.200?L frozen serum reserved at-80?was used to prepare the DNA and qPCR was performed.The free DNA concentration and the ratio of Alu247/115 in serum were calculated according to the standard curve.ELISA was used to detect the HE4 in serum of research object.The ROC curves were drawn to analyze the sensitivity and specificity.Results:1 To draw the q PCR standard curve of human genome DNA amplified with Alu115 and Alu247 primersThe purchased human genomic DNA were diluted 10 times series(10⁴-10^-3 ng/mL)and used as templates,then qPCR was performed with Alu115 and Alu247 primer respectively.The standard curves were drawn.2 The absolute concentration and integrity index of serum DNA in normal controls and ovarian patientsNormal controls:The median?IQR 25-75?ctDNA consentration?Alu115?of the 103 normal controls was 346.84?IQR216.83-479.58?ng/mL.The median serum DNA integrity index?ALU247/115?was 0.477?IQR0.424-0.505?.Ovarian cancer:The median?IQR 25-75?ctDNA consentration?Alu115?of the 119 ovarian cancers was 769.64?488.32-1035.30?ng/mL.The median serum DNA integrity index?ALU247/115?was 0.646?0.529-0.828?.The ALU115 and ALU247/115 of serum DNA in ovarian cancer group was significantly higher than that in normal controls.3 Diagnostic value of serum ALU115,ALU247/115 in primary ovarian cancer patientsAlu115:The area under the ROC curve?AUC?represents the difference between ovarian cancer and normal control.The AUC for distinguishing primary ovarian cancer patients from normal controls by ALU115 was 0.809?95%CI:0.750–0.868?.Youden index was maximal when the concentration of ct-DNA was 551.62 ng/mL,and therefore it was defined as the cutoff value.At this cutoff value,the sensitivity was 67.2%at a specificity of 90.3%,the accuracy was 77.9%,the positive predictive value was 88.9%and the negative predictive value was 70.5%.Alu247/115:The AUC of serum ALU247/115 was 0.714?95%CI:0.648–0.780?.When 0.513 was defined as the cutoff value,the sensitivity was73.1%at a specificity of 87.4%,and the accuracy,positive predictive value and negative predictive value were 87.0%,96.20%and 73.8%,respectively.4 Quantitative detection of HE4 and the diagnosis value for primary ovarian cancer patientsThe serum HE4 of ovarian cancer and control group was detected by the two-antibody sandwich ELISA method.The AUC of serum HE4 was0.806(95%CI:0.749-0.862.When 226.5 pmol/L was defined as the cutoff value,the sensitivity was54.6%at a specificity of 94.2%,and the accuracy,positive predictive value and negative predictive value were 73.0%,52.0%and 64.2%,respectively.5 Detection of ALU115 and ALU247/115 in combination with HE4Compared with the diagnostic efficiency of ALU115,ALU247/115 or HE4 alone,combined detection improved the diagnostic efficiency of primary ovarian cancer to some extent.Conclusions:1 Ovarian cancer serum free Alu115DNA and alu247/115 were signi-ficantly higher than those in the healthy control group,and they were useful for the diagnosis of ovarian cancer;2 Serum free Alu115DNA and alu247/115 were associated with age,clinical stage and lymph node metastasis of ovarian cancer patients;3 The combined detection of Alu115DNA,Alu247/115 with HE4 was helpful for the diagnosis of ovarian cancer.