| Context: Circulating cell free DNA(ccfDNA),a short extracellular DNA,is shed into the bloodstream from cells apoptosis or necrosis.In patients with cancer,a proportion of cfDNA is directly derived from tumor cells,which contains molecular alterations of tumor cells,such as the genetic and epigenetic mutations.And the concentration of ccfDNA is much higher than healthy people.Therefore,ccfDNA has generated considerable attention as an ideal biomarker of oncology.Objective: To explore the value of quantitative ccfDNA and methylation statue in assisting with diagnosis of epithelial ovarian cancer(EOC).Methods: Peripheral blood samples were obtained from EOC patients and cancerfree females as the control at Renji Hospital,School of Medicine,Shanghai Jiaotong University between April 2016 and January 2017.ccfDNA was extracted by the QIAamp Circulating Nucleic Acid Kit,and measured by the Qubit 3.0 fluorimeter,and then bisulfite,library construction,target enrichment and next-generation sequencing(NGS).To explore the value of ccfDNA level in diagnosis and the association of clinical characteristics of EOC patients,T-test,Pearson Correlation Coefficient-test and Spearman Correlation Coefficient-test were used.Wilcoxon Rank Sum test was used to screen the different methylation regions between EOC patients and the controls,early and advanced EOC patients,lymph node metastasis and non-metastasis.To validate the collective prediction power of the selected DM markers,a regularized logistic regression model was constructed for plasma samples of EOC patients.Results:(1)The ccfDNA concentration of patients with EOC was significantly higher than the controls(P<0.001).The area of ROC curve is 0.755(P<0.001).(2)The ccfDNA concentration was correlated with the logarithm of tumor size in EOC(r=0.78,P<0.001).There were no statistically significant differences between ccfDNA concentration and age,histopathological type,FIGO stage,lymph node metastasis,single or bilateral tumor,and CA125 level and ki67 expression in EOC patients(P> 0.05).(3)A total of 128 different methylation markers of EOC were confirmed.The AUC of the logistic regression model was more than 90%(P <0.001).And the average of AUC in robustness was up to 99%(P <0.05).(4)7 different methylation regions showed significant difference between early and advanced patients with EOC(P <0.05),and 21 different methylation regions exhibited correlations with lymphatic metastasis(P <0.05).Conclusion: The concentration and methylation regions of ccfDNA contributes to the diagnosis of patients with EOC,reflects the tumor burden,and evaluates staging and tumor metastasis. |
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