| Background:The incidence of prostate cancer(PCa)in China has been on a significant upward trend,and it is the fastest growing tumor in terms of incidence and mortality in China.Early PCa is limited to the capsule,which can be cured by radical surgery or radiotherapy and have a 5-year survival rate close to 100%;but when the tumor spreads outside the capsule or distant metastasis occurs,the treatment of the tumor is quite challenging,and the prognosis is very poor.Different from Western countries,most of the PCa patients in China have advanced tumors upon detection,and local progression or distant metastasis may occur.Therefore,early diagnosis of PCa is of great significance for the treatment,prognosis and survival of PCa patients.Prostate specific antigen(PSA)plays a very important role in the early diagnosis of prostate cancer.However,PSA has several limitations: 1)PSA is organ-specific but not cancer-specific.2)PSA is particularly poorly specific in the "diagnostic gray-area" patient population.3)the sensitivity in diagnosing PCa is still not adequate and about 15% of patients with PCa will be left undiagnosed.Therefore,there is an urgent need for high-sensitivity and specific molecular markers to improve the diagnostic accuracy of prostate cancer,reduce over-diagnosis and treatment,reduce social and economic burden,clarify patient molecular typing,and improve patient prognosis.Latest researches have demonstrated important role of exosomes secreted by tumor cells in the pathogenesis and development of different malignancies.Exosomes,actively secreted by cells,composed of lipid bilayers and encapsulated extracellular vesicles with genetic material,which determines its biological stability in morphology.Functionally,exosomes target specific receptor cells through small molecules on their surface and transmit their genetic material,and ultimately regulate the physiological or pathological status of the recipient cells.With their unique natural characteristics,exosomes have become a focus of researches on molecular diagnosis of cancers,which is also a major discovery in the field of liquid biopsy to break through the "bottleneck" of stability limitations.However,as of now,no systematic reports of exosome mRNA for early diagnosis of tumors have been reported.Purpose:The main research objective of this study is to screen PCa-related mRNAs by serum exosomal RNA sequencing of patients undergoing prostate biopsy,and to evaluate their role in the early diagnosis of PCa.Further,we aim to find new non-invasive markers of PCa in order to avoid unnecessary puncture and improve early-diagnosis efficiency.Thirdly,this study aims to provide relevant theoretical basis for the development of the novel prognostic cancer marker kit for prostate cancer.Methods:Sequencing technology was used in this study to sequence the serum exosomal RNA of 50 patients undergoing prostate biopsy.Data were analyzed and Lasso regression was used to screen out the most diagnostically relevant genes,and the distribution of these genes was then identified.Targeted probes are used to ensure the stability of detection,and the detection method of exosomal RNA was optimized.For genes that can be effectively detected,serum samples from 267 patients undergoing prostate puncture in Changhai Hospital were used to detect the diagnostic ability of exosome molecules in differentiating cancer and BPH.Univariate logisitic regression was used to identify independent risk factors in different subgroups of patients.The diagnostic power and efficiency of exosomal RNA in different groups of patients were compared using PSA.A diagnostic model was constructed using multivariate logistic regression,investigating whether exosomal RNA can improve diagnostic efficiency in different groups of patients.DCA curve was used to calculate the patient's net benefit,and nomogram was used to quickly calculate the cancer risk in patients undergoing prostate biopsy.Results:Through the analysis of the sequencing data,we found that genes in 9 exosomes have the potential to be a diagnostic marker for prostate cancer.After fragment distribution analysis,specific probes were synthesized and verified,which had the capability to detect 4 genes(MAX,IL32,STK4 and PDGFA).Clinical samples were used to detect the above genes and we found that MAX,IL32,and PDGFA were different in prostate cancer and hyperplastic patients.Diagnostic efficiency analysis of these three genes showed that the use of exosomal RNA alone did not differ significantly from the diagnostic efficiency of PSA in the general population and those with gray-area PSA level.Meanwhile,the diagnostic efficiency of combined exosomal RNA was not significantly different from PSA in the overall population(p=0.996).However,exosomal RNA has significantly better diagnostic efficiency in patients with gray area PSA levels(4-10 ng/ml)compared to PSA(AUC:0.718 vs 0.542,p=0.0262),indicating a potential diagnostic role of exosomal RNA in patients with gray area PSA levels.Comparing the comprehensive model containing exosomal RNA based on multivariate regression analysis with the basic diagnosis model,the AUC of the comprehensive model was significantly higher in the whole puncture population(0.872 vs 0.809,p=0.0189),the AUC in patients with PSA levels of 4-10 ng/ml was also significantly increased(0.8181 vs 0.7058,p=0.0348),and similar results were found in patients with PSA levels of 4-20 ng/ml(0.8518 vs 0.7738,p=0.0273).Thus,the diagnostic model of circulating exosomal RNA significantly enhanced the diagnostic efficiency of the basic model and improve the diagnostic efficiency of patients with gray-area PSA level.Meanwhile,by analysis of the number of missed patients with PSA levels of 4-10ng/ml with a threshold probability of 15%,we found that the number of unnecessary biopsy could be more significantly reduced(28 vs 12)using this comprehensive model of exosomal RNA in patients with GS>7.DCA curve analysis showed that patients with gray-area PSA level could have the greatest net benefit for using a comprehensive model under a threshold probability of 20%-40%.The nomogram based on the comprehensive model can be helpful for a rapid clinical decision-making based on the risk of prostate cancer in patients undergoing prostate biopsy.Conclusion:Based on the serum sequencing data of prostate patients undergoing prostate biopsy,this study screened prostate cancer-related exosome genes MAX,IL32,and PDGFA and confirmed a differentiating capability between prostate cancer and benign prostatic hyperplasia,which might be a potential marker for early diagnosis of prostate cancer.The use of the diagnosis of biopsy patients,especially PSA gray area patients,can significantly improve the diagnostic efficiency in patients with gray-area PSA level,thereby avoiding unnecessary biopsy and reducing the economic and social burden.This study also provides a theoretical basis for the development of early diagnostic kits for prostate cancer. |
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