The Application Of Prostate Specific Antigen And Its Fucosylation In The Differentiation Of Aggressive From Non-aggressive Prostate Cancer

Background:Prostate cancer(PCa)is one of the most common malignancies.Gleason Score(GS)is the "gold standard" for PCa invasiveness.In recent years,there have been many studies about markers for differentiation aggressive from non-aggressive PCa.Prostate specifitic antigen(PSA)is the most commonly used marker for early diagnosis of PCa.However,the role of PSA in differentiating aggressive from non-aggressive PCa has not been fully verified.Fucosylation of glycoproteins has been reported to play vital roles in tumor development.Lectin is a protein that been widely used in the enrichment of fucosylated proteins.Objective:1.To analyze and compare the PSA level in cells,serum and urine of aggressive and non-aggressive PCa.2.To develop two lectin-based immunoassays for selection of fucosylated PSA followed by clinical PSA immunoassay,and its performance was evaluated.3.The level of fucosylated PSA in aggressive and non-aggressive PCa sample were detected by lectin-based clinical PSA immunoassay.Exploring the role of fucosylated PSA in the differentiation and diagnosis of aggressive and non-aggressive PCa.Methods:1.The aggressive and non-aggressive PCa cell models were established by transfection of FUT8 gene,then the models were evaluated.The FUT8 gene was transfected into LNCap PCa cell line,and the LNCap COT cell models and LNCap FUT8 cell models were constructed to represent the aggressive and non-aggressive PCa cells,respectively.Western blot was used to detect the expression of FUT8 in LNCap FUT8 cells and LNCap COT cells.Then Western blot was further used to qualitatively detect the PSA levels in LNCap COT and LNCap FUT8 cells and the culture medium.Furthermore,chemiluminescence immunoassay was used to detect the level of PSA between these two groups.2.Serum and urine samples of PCa patients were collected to evaluate their PSA levels.Serum samples from 24 PCa patients were collected.Among these serum samples,33.3%cases(n=8)were Gleason 6,33.3%(n=8)were Gleason 7,12.5%(n=3)were Gleason 8,and 20.8%(n=5)were Gleason 9.The serum PSA levels in PCa patients with different Gleason scores were determined by chemiluminescence immunoassay.Urine samples from 146 PCa patients were collected,including 47.9%(n=70)cases of Gleason 6,19.2%(n=28)cases of Gleason 8,28.1%(n=41)cases of Gleason 9 and 4.8%(n=7)cases of Gleason 10.The urine PSA levels of each Gleason score group in PCa patients were determined by chemiluminescence immunoassay.3.Development of two lectin-based immunoassays for selection of fucosylated PSA followed by clinical PSA immunoassay.This glycoproteomic strategy is based on agarose gel-conjugated lectin-specific selection for fucosylated glycoproteins followed by PSA measurement using immunoassay.The performance of this assay was further evaluated.4.To evaluate fucosylated PSA as a tumor marker and its potential application in the diagnosis of aggressive and non-aggressive PCa.The fucosylated PSA in aggressive and non-aggressive PCa samples were enriched by agarose gel-conjugated lectin,and its levels were evaluated to compare the differences.Results:1.The LNCap PCa cells transfected with FUT8 gene were detected to have higher FUT8 expression than LNCap COT cells which did not have FUT8 transfected,which showed that the models of aggressive PCa cells and non-aggressive PCa cells were successfully established.PSA levels in aggressive PCa cells and medium were significantly lower than that in non-aggressive PCa.2.Serum PSA levels of Gleason 8 and Gleason 9 were significantly higher than those in the Gleason 6 group.However,there was no significant difference in serum PSA levels between group Gleason 6 and Gleason 7,neither when compared with group Gleason 7 and Gleason 8+Gleason 9.However,we found the PSA level was higher in group Gleason(4+3)+Gleason 8,Gleason 9 than group Gleason(3+4)+Gleason 6.The AUC of receiver operating characteristic(ROC)curves analysis in the differentiating Gleason pattern 7(4+3)and above with Gleason patterns 6 and 7(3+4)was 0.881(p=0.002),which was statistically significant.3.In urine samples,PSA level in the Gleason 6 group was significantly higher than that in the Gleason 8,Gleason 9,Gleason 10 group,statistically significant difference of PSA level was found between the two groups.The AUC was 0.739(p=0.002),which was statistically significant.4.LCA and AAL enriched fucosylated PSA level showed a linear relationship with the total PSA level(Y=0.2105X+0.0910,R2=0.9985;Y=0.0543X-0.0182,R2=0.9973)within a range of 0-80 ng/mL.The ratio of fucosylated PSA enriched by LCA and AAL(fucosylated PSA level/total PSA level)were 21.05%,5.43%.The coefficient of variation(CV)values for low,median,high of LCA enriched fucosylated PSA combined with chemiluminescence method was less than 10%and bias<10%.The coefficient of variation(CV)values for low,median,high of AAL enriched fucosylated PSA combined with chemiluminescence method were less than 11%and bias<11%.The recovery rate was 80%-110%,no obvious cross-reaction with Prostatic acid phosphatase(PAP).5.There was no statistically significant difference of fucosylated PSA level that enriched by AAL in the cells and medium between aggressive PCa and non-aggressive PCa.However,the ratio of fucosylated PSA and total PSA in non-aggressive PCa cells and medium was significantly higher than that in aggressive PCa.The level of fucosylated PSA enriched by LCA in non-aggressive PCa cells and medium was higher than that of aggressive PCa.However,there was no significant difference in the ratio of fucosylated PSA between these two groups.6.After enriched by AAL,the serum fucosylated PSA level and ratio of fucosylated PSA in the Gleason 8 and Gleason 9 groups were higher than those in the Gleason 6 and Gleason 7 groups.There was a statistical difference of level of fucosylated PSA and the ratio of fucosylated PSA between group Gleason 6,Gleason 7,Gleason 8 and group Gleason 9.The serum fucosylated PSA level and ratio of fucosylated PSA and total PSA in the Gleason(4+3)+Gleason 8+Gleason 9 group were higher than those in the Gleason(3+4)+Gleason 6 group.After enriched by LCA,the serum fucosylated PSA level in group Gleason(4+3)+Gleason 8+Gleason 9 was higher than that in group Gleason(3+4)+Gleason 6.But there was no significant difference of fucosylated PSA ratio between the two groups.The AUC of the fucosylated PSA enriched by AAL,ratio of fucosylated PSA enriched by AAL,fucosylated PSA enriched by LCA and ratio of fucosylated PSA enriched by LCA were 0.909,0.822,0.839 and 0.594,respectively.7.After the fucosylated PSA enriched by AAL and LCA in urine,we didn’t find significant difference of fucosylated PSA level and fucosylated PSA ratio between group Gleason 8 and Gleason 9 and group Gleason 6 and Gleason 7.Also,there was no statistical difference of fucosylated PSA level and fucosylated PSA ratio among group Gleason 6,Gleason 7.Gleason 8 and Gleason 9.Conclusion:1.The aggressive and non-aggressive PCa cell models were successfully established in our study.The PSA level in non-aggressive PCa cells and urine was higher than that in aggressive PCa,while the PSA level in the aggressive PCa serum was higher than that nrnon-aggressive PCa.2.We successfully established two lectin-based(LCA,AAL)immunoassays for selection of fucosylated PSA followed by clinical PSA immunoassay.3.The established method was used to detect PCa samples,and it was found that fucosylated PSA-AAL,%fucosylated PSA-AAL,fucosylated PSA-LCA could improve the predictive power and might provide additional information in the differentiation of Gleason pattern of 7(4+3)and above with the Gleason pattern 6 and 7(3+4).Among these,fucosylated PSA-AAL achieved a better performance.