TERT-based CTC Detection For Early Diagnosis And Metastatic Prediction Of Prostate Cancer

Background:Prostate cancer is one of the most common malignant tumors in men,and the incidence and mortality of prostate cancer are increasing in China.However,There remain two difficulties in the diagnosis of prostate cancer:1.The diagnostic specificity of the "PSA diagnostic grey area(4-10 ng/mL)" is low;2.Lack of early effective warning methods for prostate cancer metastasis.Circulating tumor cells(CTCs)can enter the blood in the early stage of malignant tumors,and CTCs in peripheral blood are regarded as "seeds" of metastasis.Accordingly,we used a method developed previously,that is herpes simplex virus type I mediated telomerase reverse transcriptase(TERT)activity tracing method for prostate cancer CTC detection.In this study,we evaluated the diagnostic ability of TERTactive CTCs in early-stage prostate cancer,especially in the "PSA diagnostic gray area".Then,we explored the potential of TERT and PSMA dual-positive CTCs in early warning of prostate cancer metastasis.Based on above,we finally established a novel CTC detection technique,named PSA-based CTC detection(PBCD),which could be applied on the diagnosis of prostate cancer independently.Method:In vitro simulation experiments on TERT high expressed prostate cancer cell lines were conducted to verify the reliability of TBCD and the reliability of CTCs was tested by FISH and flow imaging.A double-blind,prospective clinical study was designed to evaluate the diagnostic efficacy of TBCD.The patients suspected to have prostate cancer were enrolled and randomly divided into the training group and validation group receiving CTC detection.ROC curve were drawn to calculate the AUC value.GO enrichment analysis was performed to analyze the correlation between prostate cancer CTCs with high PSMA expression and metastasis;TBCD was applied to detect "TERT and PSMA double-positive CTCs" and test its association with tumor in metastatic tumor group and local tumor group in our cohort.Finally,CRISPR-Cas9 technology and recombination repair technology were used to construct PBCD;the reliability of PBCD was verified by in vitro simulation experiments on PSA high expression prostate cancer cell lines and its diagnostic efficacy was preliminarily evaluated in patients with suspected prostate cancer.Results:1.In vitro simulation experiments showed that the TBCD had good repeatability,the recovery rate was greater than 70%,the inter-assay and intra-assay CV values were less than 20%,the minimum detection limit was less than 5 added quality control cells,and the linear regression coefficient was>90%;CTCs originated from prostate cancer were identified by flow imaging,and its genotype of PTEN loss-of-heterozygosity mutation were confirmed by FISH.2.The results of the clinical trial showed that the AUC of TBCD in the training group was 0.842 with a diagnostic threshold of "≥2 CTCs/4 mL peripheral blood";the AUC in the validation group was 0.789;In the range of "4≤PSA<20 ng/mL" and "4≤PSA<10 ng/mL",the AUC was 0.811 and 0.825 respectively,and the diagnostic sensitivity and specificity were higher than 80%,which was significantly better than PSA(p<0.05);3.In the gray area of PSA,the AUC could be further improved to 0.837 in the "CTC+PSA combined model",and the DCA curve suggested the combined model had better clinical application potential;4.The results of GO enrichment analysis indicated that CTCs with high expression of PSMA were associated with prostate cancer metastasis;among the 27 metastatic and 44 local prostate cancer patients,13(48.15%)and 10(22.72%)had "PSMA and TERT double-positive cells" respectively,and the difference was statistically significant(p<0.05);5.The new prostate cancer CTC detection method PBCD was successfully established.In the preliminary evaluation of CTC detection ability,its AUC was 0.97,with 76.5%sensitivity and 100%specificity,which were similar to TBCD.Conclusions:1.TBCD can be applied to the detection of prostate cancer CTC;2.TBCD can assist PSA to improve the diagnostic ability for early prostate cancer;3."PSMA and TERT double-positive CTCs" have great potential in early warning and prediction of prostate cancer metastasis;4.PBCD,a novel prostate cancer CTC detection method have great potential in prostate cancer-specific diagnosis.