The Application Value Of Serum Exosome Membrane Protein GPC-1 In The Diagnosis Of Breast Cancer

Part one The extraction and identification of serum exosomesObjective:To establish the extraction method of serum exosome and to identify the exosomes in the precipitations of the subjects.Methods:The subjects were 66 patients with newly diagnosed breast mass,in which were 18 with benign breast diseases and 48 with breast cancer,meanwhile 17 healthy people were included as health control.1 A method was established to extract exosomes from serum and exosomes were extracted from serum samples of subjects from healthy control,breast benign disease group,and breast cancer group.2 Transmission electron microscope was used to observe the shape and size of exosomes in the precipitations.3 Western blot was used to detect the expression of CD9 in the precipitations of the subjects.Results:The method of 50%PBS-NaCl solution of polyethylene glycol 8000 to extract exosomes from serum was established;the exosome observed by transmission electron microscope were considered typical,its diameter was about 100nm,and CD9 was highly expressed in the precipitations of the subjects.Part two The application value of GPC-1 level of serum exosome in the diagnosis of breast cancerObjective:To detect the content of serum exosome membrane protein GPC-1 in healthy people,patients with benign breast disease and breast cancer,and explore its application value in the diagnosis of breast cancer.Methods:1 Western blot was used to detect the expression of GPC-1 in the precipitations of the subjects,to see if there were any differences between three groups.2 The application value of serological markers,imaging results and GPC-1 expression level in the diagnosis of breast cancer was compared with the pathological results as gold standard.Results:1.The expression level of GPC-1 in healthy control group was(median 0.635,quartile range 0.145),in benign disease group the level was(0.661,0.169),breast cancer group(1.065,0.501).Kruskal-Wallis H test was used in comparing the results,it was found that the expression level of GPC-1 in three groups was not the same(H=32.051,P<0.05),and that in breast cancer group was significantly higher than that in healthy control group and benign disease group.There was a positive correlation between GPC-1 level in breast cancer group before treatment and tumor staging(r_s=0.536,P<0.05),the later the tumor stage,the higher the content of GPC-1.Between the GPC-1positive and negative groups,the difference of cancer staging,node infiltrating and the expression of Ki-67 was statistically significant,patients with positive GPC-1 level usually have later tumor staging,node infiltration and high expression of Ki-67.After treatment,the level of GPC-1 in breast cancer group was(median 0.844,quartile range 0.195),which was decreased compared with that before treatment using Wilcoxon signed-rank test(Z=856.0,P=0.006).2.The GPC-1 expression level was used to draw the ROC curve of breast cancer.The diagnostic boundary value was 0.961,the sensitivity and specificity of diagnosing breast cancer were 72.9%and 97.1%respectively,the area under the curve was 0.900(95%CI:0.833-0.967).The sensitivity of serological tumor marker detection in breast cancer dignosing was 22.9%(11/48),the specificity was 94.4%(17/18),Kappa=0.107;and the ultrasonography detecting sensitivity 97.9%(47/48),specificity 16.7%(3/18),Kappa=0.193;the molybdenum target X-ray examination sensitivity 91.7%(44/48),specificity 27.8%(5/18),Kappa value=0.230;the sensitivity of GPC-1 was 66.7%(32/48),the specificity was 94.4%(17/18),Kappa=0.485.Conclusions:1.The Polyethylene glycol 8000 precipitation method can be used to extract exosomes from serum samples.2.The expression level of serum exosome membrane protein GPC-1in breast cancer patients is higher than that in healthy controls and benign breast diseases.The content of GPC-1 is positively correlated with tumor stage and decreases after treatment.Compared with GPC-1 negative patients,GPC-1 positive patients are prone to lymph node metastasis,and the clinical stages are late,and the proliferation of tumor cells is higher.3.The serum exosome membrane protein GPC-1 level is highly sensitive and specific in the diagnosis of breast cancer,and has the potential to be a new serological marker for the diagnosis of breast cancer.