| Objective : The overall purpose of this study is to detect circulating cell-free DNA(cfDNA)in serum and explore the value of its content and integrity index(the ratio of long DNA content to short DNA content)in the diagnosis and treatment of gastric cancer.To study the diagnostic value of single detection of cfDNA content,integrity and combined detection of tumor markers for gastric cancer,and find out the indicators with high sensitivity and specificity.Method:A case-control study was conducted in this research?In this study,30 patients with gastric cancer diagnosed for the first time in the Affiliated Hospital of Beihua University from June 2018 to November2018(11 of whom underwent gastrectomy).30 patients with gastric cancer were first diagnosed in the Affiliated Hospital of Beihua University from June 2018 to November 2018(11 of them underwent gastrectomy);30 patients with benign gastric diseases were selected in the same period;30 healthy people in the same period were selected in the control group;all cases were diagnosed by gastroscopy and pathology.Serum samples were collected from all subjects(blood samples were collected before and on the 7th day after gastric cancer surgery).The samples were frozen at-80? for reserve.Firstly,the serum cfDNA of all the subjects was extracted and frozen at-80 ? for reserve.Electrophoresis of the extracted serum cfDNA wasperformed by agarose gel electrophoresis.The genomic DNA of healthy human blood was extracted and quantified by ultraviolet spectrophotometer.The DNA was diluted into 5 concentration gradients of 1-10000 ng/ml.All the cfDNA and standard DNA were amplified by PCR using ALU-115 fragment and ALU-247 fragment as primers,and the specific content was obtained according to fluorescence quantitative results.Using the fluorescence threshold(CT value)of the standard sample in the process of PCR amplification as abscissa and logarithm(lg concentration)of the concentration as ordinate,the standard curve for the determination of serum cfDNA concentration was established,and the DNA concentration and integrity index of ALU-115 and ALU-247 fragments in different subjects were obtained.To compare the content and integrity of cfDNA in gastric cancer group,benign gastric disease group and healthy control group,and to explore the changes of serum free circulating DNA content and integrity in three groups of subjects and its diagnostic value for gastric cancer,based on the clinical and pathological data of gastric cancer patients.By comparing the changes of cfDNA content and integrity before and after operation,the value of cfDNA in the treatment of gastric cancer was discussed.Finally,ROC curves are drawn for sensitivity and specificity analysis of single index and joint detection results.Results:1.Draw the standard concentration curves of ALU-115 and ALU-247 in human whole blood genome.2.The concentration and integrity index of ALU-115 and ALU-247 DNA fragments.In gastric cancer group,the median content of Alu-115 fragment(quartile spacing)was 500.151 ng/ml(IQR422.797-916.144)ng/ml;the median content of Alu-247 fragment(quartile spacing)was 106.089ng/ml(IQR63.604-188.175)ng/ml.In benign gastric disease group,the median content of Alu-115fragment(quartile spacing)was 164.926 ng/ml(IQR120.304-291.394)ng/ml;the median content of Alu-247 fragment(quartile spacing)was43.878 ng/ml(IQR21.228-77.192)ng/ml.In healthy group:The median content of Alu-115 fragment(quartile spacing)was 116.357 ng/ml(IQR53.440-252.851)ng/ml and that of Alu-247 fragment(quartile spacing)was 26.608 ng/ml(IQR10.850-67.093)ng/ml.The median content of Alu-115 fragment in gastric cancer group was significantly different from that in benign gastric disease group and healthy group(Z=-5.13,P=0.000;Z=-6.15,P=0.000).There was no significant difference in the median content of Alu-115 fragment between benign gastric disease group and healthy group(Z=-1.818,P=0.069).The median content of Alu-247 fragment in gastric cancer group was significantly different from that in benign gastric disease group and healthy group(Z=-3.622,P=0.000;Z=-5.382,P=0.000),while there was no significant difference in the median content of Alu-247 fragment between benign gastric disease group and healthy group(Z=-1.892,P=0.058).The median integrity index was 0.166(IQR0.137-0.303)and 0.260(IQR0.144-0.388)for gastric benign diseases and 0.253(IQR0.169-0.369)for healthy people.There was no significant difference in integrity index between 0.253(IQR0.169-0.369)in gastric cancer group,benign gastric disease group and healthy population group(Z values were-1.575,-1.626,-1.33,P > 0.05).3.The relationship between serum cfDNA content and integrity and Clinicopathology in gastric cancer group.Serum levels of cfDNA Alu-115 and Alu-247 fragments weresignificantly different from TNM staging in patients with gastric cancer(KW=11.709,P=0.008;KW=10.085,P=0.018).There was no significant difference in integrity index between patients with gastric cancer and TNM staging.There was no significant difference in serum cfDNA concentration and integrity between patients with gastric cancer and clinicopathological characteristics such as sex,age and smoking history,but no significant difference in HP infection,histopathological classification,differentiation degree,lymph node metastasis and distant metastasis.4.The value of serum cfDNA content and integrity in the treatment of gastric cancer.The median content of Alu-115 fragment was 496.569 ng/ml in preoperative group(IQR427.685-920.975)ng/ml,215.465 ng/ml in postoperative group(IQR162.017-843.187)ng/ml.There was significant difference before and after operation(Z=-2.003,P=0.045).The median content of Alu-247 fragment was 133.240 ng/ml in preoperative group(IQR54.002-244.823)ng/ml,51.698 ng/ml in postoperative group(IQR32.810-134.073)ng/ml.There was no significant difference before and after operation(Z=-1.609,P=0.108).The median integrity index was0.201(IQR0.145-0.299)in the preoperative group and 0.189(IQR0.149-0.411)in the post-operative group.There was no significant difference in the content of Alu-247 fragments and the integrity index before and after operation(Z=-0.296,P=0.767),but there was a decreasing trend after operation.5.Comparison of sensitivity and specificity of combined detection of ALU-115,integrity and tumor marker CA199 in the diagnosis of gastric cancer.The area under ROC curve of ALU-115,integrity and tumor marker CA199 could reach 0.976.The sensitivity and specificity of ALU-115 andCA199 were 90% and 96.3% respectively.Compared with single detection,combined detection has more diagnostic value for gastric cancer.Conclusions:1.The expression of cfDNA in serum of patients with gastric cancer increased significantly,and the expression of cfDNA decreased significantly after gastric cancer operation.This indicates that cfDNA content is expected to be a tumor marker for the diagnosis of gastric cancer and has clinical value for the evaluation of curative effect of gastric cancer.2.The sensitivity of combined detection of cfDNA content,integrity and tumor markers in the diagnosis of gastric cancer is higher than that of single detection.The specificity of combined detection of cfDNA content,integrity and tumor markers in the diagnosis of gastric cancer is higher than that of single detection. |
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