Application Of Urinary Exosome AMACR Test For Early Diagnosis Of Prostate Cancer

Background and ObjectiveProstate cancer(PCa)is a serious threat to men’s health and is one of the leading causes of death in men worldwide.Prostate cancer has no obvious symptoms in the early stage,which makes it difficult to diagnose,and since prostate cancer screening is not yet popular,many patients have already developed to high-risk stage or even metastasis by the time of diagnosis,with poor prognosis.Therefore,early diagnosis of prostate cancer is of great clinical and social value.As the most recognized and widely used biomarker for the diagnosis of prostate cancer,PSA plays an important role in clinical screening.PSA plays an important role in clinical screening.However,due to the poor specificity of PSA,there is an urgent need to explore new methods and techniques for prostate cancer screening and diagnosis in order to improve the predictive efficacy.Liquid biopsy is considered as a tool to identify alternative biomarkers.Urine is absolutely non-invasive and is the most readily available of the clinical samples.Several exosomal proteins in urine have shown high sensitivity and specificity in PCa,and exosomal proteins in urine have not been fully explored as an easily collected and non-invasive source of cancer biomarkers.In this study,we propose to screen for proteins that are significantly different within urinederived exosomes from prostate cancer and normal prostate hyperplasia patients after rectal examination by 4D Label-free protein profiling and perform bioinformatic analysis to mine potential prostate cancer predictors.To explore the expression of AMACR in prostate cancer tissues,prostate cancer cells and cell supernatant-derived exosomes.To further identify and multicenter validate urinary exosomal AMACR as a novel biomarker to improve the detection of prostate cancer(PCa)and clinically significant PCa(Gleason score≥7)at initial biopsy.Methods1.Part I:Screening for proteins that are significantly different from those in urine-derived exocrine bodies after rectal examination in patients with prostate cancer and normal prostate enlargement by 4D Label-free protein profiling and bioinformatic analysis.2.Part II:Detection of AMACR expression in prostate cancer and normal prostate tissues by immunohistochemistry;detection of AMACR mRNA expression in prostate cell lines and normal prostate epithelial cells by RT-PCR;detection of AMACR expression in supernatant exosomes of prostate cell lines and normal prostate epithelial cells by WB.3.part III:Patients who underwent prostate puncture at our center from December 2017 to December 2019 were included according to established inclusion and exclusion criteria,and the first urine sample was collected after rectal examinations.Urinary exosomes were purified and urinary exosome AMACR(UE-A)was measured by enzyme-linked immunosorbent assay(ELISA).The diagnostic performance of UE-A was assessed by receiver operating characteristic(ROC)analysis,decision curve analysis(DCA)and waterfall plot.Results1.part I:protein profiling assay,73 differentially expressed proteins were identified,of which 50 were significantly up-regulated in the urine-derived exosomes of prostate cancer patients,with ALDH6A1,AR,AMACR,PCBD1 and GCDH being the top five proteins significantly up-regulated.Bioinformatics analysis of the significantly elevated proteins in exocytosis revealed that the differentially expressed proteins were highly enriched in cellular processes of amino acid catabolism,small molecule catabolism and organic acid catabolism;highly enriched in mitochondrial matrix,peroxisomes and microsomal environment;and highly enriched in the molecular biological functions of coenzyme binding.The results of KEGG pathway enrichment analysis suggest that the upregulated differential proteins are highly enriched in the degradation of valine,leucine and isoleucine.2.Part Ⅱ:Immunohistochemical results showed that AMACR was highly expressed in prostate cancer tissues compared to normal prostate tissues and positively correlated with and with Gleason score;RT-PCR assay suggested that AMACR mRNA was highly expressed in prostate cell lines,with the highest expression level in PC3 cells;WB experiments suggested that AMACR was highly expressed in prostate cell lines compared to normal prostate epithelial cells.AMACR expression was significantly higher in cell supernatant exosomes of prostate cell lines compared to normal prostate epithelial cells.3.part Ⅲ:A total of 612 patients with elevated PSA were included,divided into a training cohort(before 2018.12)and a validation cohort(after 2018.12),with a sample size of 306 cases in each cohort.expression of AMACR was significantly higher in PCa and clinically significant PCa(csPCa)than in BPH and non aggressive disease(nsPCa)(p<0.001).ue-A performed well in distinguishing PCa from BPH or BPH plus nsPCa with an area under the ROC curve(AUC)of 0.750,0.829,respectively.ue-A’s performance in a multicenter cohort of patients was further validated with an AUC of 0.747 for detection of PCa and 0.794 for detection of csPCa.clinical utility assessed by DCA showed that patients using UE-A in both the training and validation cohorts had superior benefit over PSA and f/t PSA in all threshold probabilities.ConclusionBased on the protein profile results,we screened urinary exosomes for AMACR as a possible new biomarker for prostate cancer diagnosis;AMACR was significantly elevated in prostate tissue,prostate cancer cell lines and prostate cancer cell supernatant-derived exosomes;we demonstrated the advantage of UE-A in early diagnosis of PCa and csPCa.ue-A could be a novel non-invasive biomarker to improve the efficacy of detection of PCa and csPCa.