Prostate cancer is the most common malignant tumor of the male reproductive system and the fifth leading cause of cancer death in men. In 2020, there were 1.414 million new cases and 375,000 deaths worldwide. China reported 115,000 new cases and 51,000 deaths. Although China's morbidity and mortality rates are low and the growth rate is fast, the 5-year survival rate is only 69.2%, which is more than 80% lower than that in Europe and the United States. May be due to a poor prognosis for late diagnosis. Early diagnosis of aggressive prostate cancer is required to improve survival.
Early diagnosis of prostate cancer (PCa) currently relies on PSA testing and biopsy, but there are problems with insufficient sensitivity and specificity, especially in the "gray zone". This can lead to overdiagnosis and treatment. Researchers seek biomarkers in urine, such as DNA, RNA, proteins, and exosomes, to improve noninvasive diagnostic methods. PSA is widely used in prostate cancer screening, but it has the problems of high sensitivity and low specificity, and is susceptible to other factors. PSA 4~10μg/L is a gray area, and it is difficult to distinguish benign diseases. PSA was within this range in 33% of patients with metastatic PCa. 96% of men did not suffer from PCa at the level of 2.0~10μg/L. The guidelines of the Chinese Medical Association recommend the use of fPSA/tPSA, PSAD, PSAVE and other parameters to improve the accuracy of screening, but the application conditions and diagnostic values are controversial. This paper summarizes the relevant research progress in recent years and shares the exploration results of new non-invasive prostate cancer risk prediction indicators.
Patients may choose between surgery or radiation therapy, which may be associated with side effects. Low- and intermediate-risk patients are given priority to active surveillance (AS), and their condition is evaluated regularly with PSA, biopsy, etc. Advantages of liquid biopsy analysis of circulating tumor cells, DNA, proteins, and exosomes (EVs) as biomarkers:
(1) EVs are present in most body fluids and are more readily available;
(2) Various molecules (proteins, nucleic acids, such as mRNA and miRNA, lipids, metabolites, etc.) that represent tissue specificity;
(3) Exosomes are encapsulated with phospholipid bilayers, which are more stable than free-circulating biomarkers.
1
Urine tumor markers
1.1 PCA3
PCA3 is a long, non-coding RNA located on chromosome 9 and was discovered in 1999. It is overexpressed in prostate cancer and metastatic tissues, and the PCA3 score can be obtained by measuring the ratio of PCA3 to PSA mRNA concentration. This score is the first FDA-approved urine tumor marker test. Proussard et al. reviewed that PCA3 was better than serum PSA in diagnosing PCa, with an AUC of 0.64-0.83.
Meta-analysis (54 studies, 17,575 patients) showed that PCA3 score was superior to serum PSA in the diagnosis of PCa: sensitivity 71%, specificity 68%, diagnostic odds ratio 5.28, and pooled AUC 0.75.
Studies have shown that the combination of PCA3 and serum PSA has some value in the diagnosis of prostate cancer (PCa), mainly to help in the decision to repeat biopsy. However, its use in assessing invasive PCa and prognosis is limited. The optimal cut-off value for PCA3 score is controversial: low values increase negative predictive value; High values increase the positive predictive value; Medium scores are uncertain. Combination with other markers may address this issue.
1.2 TMPRSS2-ERG
Gene fusions are often triggered by chromosomal rearrangements, which are present in patients with prostate cancer (PCa). In 2005, Tomlins et al. discovered for the first time that TMPRSS2 in the prostate gland is fused with ETS transcription factor genes, which are involved in cellular functions such as proliferation and apoptosis. The TMPRSS2-ERG fusion gene is detected in 50% of patients with PCa and can be detected by urine. In a study of 109 patients, it had a specificity of 93% and a positive predictive value of 94%, but a sensitivity of 37%. The combination of TMPRSS2-ERG in urine and PCA3 improves diagnostic performance, increasing sensitivity from 68 to 76 percent [17]. The University of Michigan developed the MyProstateScore (MPS) model, which is based on urine and serum metrics. In a cohort study of 1125 individuals, MPS had a positive predictive AUC of 0.75 for PCa and 0.77 for predicting aggressive PCa (Gleason≥7), both of which were superior to serum PSA alone. A prospective multicenter cohort study by Sanda et al. [19] (sample size: 1077) showed that MPS scores improved the detection rate of aggressive prostate cancer (PCa) compared with serum PSA alone. When the 95% sensitivity threshold was set, the specificity of high-grade PCa detection increased from 18% to 39%, reducing unnecessary biopsies by 42%. Osoian et al. showed that the diagnosis of MPS showed high efficiency in identifying aggressive prostate cancer. When the MPS is <10, the sensitivity and negative predictive values are high (96 versus 97 percent, respectively), and unnecessary biopsies are reduced by 33% and invasive cases are missed in only 3% of cases. TMPRSS2-ERG has clinical value in predicting aggressive PCa, but its sensitivity alone is low. In combination with PCA3, diagnostic performance can be significantly improved. The successful application of MPS suggests that the combination of multiple markers may be an effective way to improve diagnostic capabilities.
1.3 MALAT1
"MALAT1, a long, non-coding RNA on chromosome 11, has been implicated in aggressiveness in a variety of cancers (e.g., hepatocellular carcinoma, lung, colorectal) and prostate cancer (PCa). Wang et al. found that in PCa patients with PSA 4~10 μg/L, the MALAT1 score of those with positive biopsy was higher than that of those with negative biopsy. MALAT1 predicts better accuracy than total PSA or free PSA/total PSA ratio. When the threshold is 25%, the unnecessary biopsy can be reduced by 30.2%~46.5% without affecting the diagnosis of invasive PCa. MALAT1 plays an important role in prostate cancer (PCa) progression and metastasis, but its specificity is lower than that of PCA3. Studies have shown that MALAT1 has high clinical value in predicting cancer progression and metastasis, and its combination with markers with higher specificity may improve diagnostic efficacy. However, the current clinical research is insufficient, and more prospective studies are needed for validation.
1.4 PSA
In 1985, Graves first discovered the presence of PSA in urine, but early studies have contradictory results and its diagnostic value is still controversial. Recent studies have shown that low expression of PSA in urine is associated with the occurrence and progression of prostate cancer (Pca). PSA is produced by acinar and duct epithelial cells, and is released into the bloodstream in large quantities when cancer progresses, and serum PSA does not accurately reflect the PSA expression of Pca tissue. The study showed that decreased PSA expression in high-grade prostate cancer (PCa) tissues was associated with an increased Gleason score and enhanced cell proliferation. In a cohort of 527 people, Occhipinti et al. found that urine PSA reflected the expression level of PSA in prostate tissue, and its negative and positive predictive values for aggressive prostate cancer (PCA) were 82.8% and 47.9%, respectively. The urine PSA AUC was 0.691, which was better than the standard predictive model (SOC) AUC of 0.621. Urine PSA binding to SOC can be increased to 0.712. Using a threshold of 0.4 reduced unnecessary biopsies by 26% and missed invasive PCA by only 2%. Loss of PSA expression is associated with PCa stage and prognosis. Urine PSA testing has the potential to distinguish PCa from benign disease and predict staging. In view of the limited current research on urine PSA, it is recommended to verify a larger sample size or establish a scoring system in combination with other markers to improve the clinical diagnostic value.
2
Tumor markers based on urine exosomes
Exosomes: 30-180 nm diameter, extracellular vesicles with lipid bilayer membranes. It is widely found in bodily fluids such as urine of mammals. Exosomes play an important role in tumor microenvironment, invasion and metastasis, and immune evasion. They carry the nucleic acids, proteins, and metabolites of the cell, reflect the characteristics of the source cell, and are stable due to the protection of the lipid bilayer, making them ideal biomarkers.
2.1 Urine exosomal tumor markers and their combined detection
ExoDx Prostate Test: The first FDA-cleared urine exosome biopsy. The EPI score was detected by PCA3, ERG and SPDEF to assist PSA screening. EPI >15.6 high risk. No prostate massage required.
Donovan et al. [37] found that the ExoDx Prostate Test had a negative predictive value of 97.5% and a positive predictive value of 37.5% for high-grade prostate cancer (PCa) in newly detected patients. Combined with the EPI score and standard operating procedure (SOC) model to improve the diagnostic value, the AUC increased from 0.67 to 0.80. The validation study showed that when the EPI score was truncated at 15.6, the diagnostic sensitivity of invasive PCa was 91.9%, the negative predictive value was 91.3%, and the AUC was 0.71. The combination of ExoDx and SOC had an AUC of 0.73 over SOC alone (AUC 0.63) and reduced the rate of invalid biopsies by 27 percent and missed invasive PCa by 8 percent [38].
Mckiernan et al. conducted a prospective cohort study of 504 patients over 50 years old, and found that the ExoDx Prostate Test combined diagnostic model had an AUC of 0.71, a sensitivity of 93% and a negative predictive value of 89% at an EPI score of 15.6, which could reduce prostate biopsy by 26%, with a total biopsy rate of 20% and a misdiagnosis rate of 7%. The latest cohort study by Mckiernan et al. (n=229) found that the ExoDx Prostate Test outperformed other models in the EPI score of patients with aggressive prostate cancer (PCa) who had a negative initial biopsy. When the cut-off value of EPI score was 15.6, the negative predictive rate was 92%, and the AUC was 0.66 (95% CI: 0.55~0.78), which could reduce unnecessary biopsies by 26% and miss the diagnosis of invasive PCa by only 2.6%. The study showed that 71.6% white and 14.4% African-United States were ethnically diverse. Evaluation of the ExoDx prostate test for Asia, especially in the Chinese population, is lacking.
2.2 尿液外泌体中的微RNA
miRNAs are non-coding single-stranded RNAs of about 22 nucleotides that regulate gene expression. Because it is related to the various functions of tumor cells, it has great potential as a tumor marker. Samsonov [43] and Foj [44] found that miRNA expression in urine exosomes of PCa patients varied, possibly due to different isolation methods. The combined detection of miR-21 and miR-375 had high diagnostic efficacy (AUC=0.872), but further clinical validation was needed. Rodríguez et al. found that the expression of five miRNAs in the urine exosomes of PCa patients was reduced, especially miR-196a-5p and miR-501-3p, which can be used for diagnosis. The research on urine exosomal miRNA as a diagnostic marker for prostate cancer (PCa) has just begun, and it is facing the problem of standardizing isolation methods. However, it has great potential to improve the early diagnosis and prognosis of PCa. Urine tumor markers have great potential in the early diagnosis of PCa, and the combination of multiple markers can improve the diagnostic effect. However, there is a lack of direct data on the comparison of emerging markers, and comparisons between studies are limited by differences in urine collection and sample handling. In addition, there are obvious differences in the genetic characteristics of PCa between European, American and Asian populations, and further large-scale clinical studies are needed to support the optimal cut-off value and diagnostic efficacy evaluation of Asian populations. Although PCa urine biopsy is limited, it provides valuable non-invasive cancer detection for early detection of invasive PCa and reduces unnecessary needle biopsy and waste of medical resources. In the future, the diagnosis and treatment mode may be changed, and large-scale prospective studies are needed to verify it.
3
Tumor markers based on urine exosomes
尿液exosomal miR-375：
miRNAs are small non-coding single-stranded RNAs with a length of 18-25 nucleotides that regulate gene expression by pairing with 3'-UTR to degrade or inhibit translation. Its aberrant expression is associated with tumors and can be used as a biomarker. miRNAs transmit messages between tumor cells through exosomes. Exosomes are membrane vesicle cells of 30~100nm, which are widely present in body fluids, containing specific proteins, mRNA and miRNA, regulating the behavior of recipient cells, and can be used as molecular markers of diseases. Studies have shown that miRNAs in urine exosomes reflect cellular origin and are suitable as non-invasive molecular markers for cancers such as prostate cancer. MiR-375 is a pancreatic-derived miRNA that is aberrantly expressed in prostate cancer.
The results showed that the expression of exosomal miR-375 in the urine of prostate cancer patients was significantly lower than that of BPH patients and healthy people (P<0.01), but there was no significant difference between BPH group and control group (P>0.05). This suggests that urine exosomal miR-375 may serve as a non-invasive diagnostic marker for prostate cancer. Although other studies have explored the potential of miR-375 in prostate cancer, its diagnostic value as a urine biomarker has not been reported.
urine CXCL16:
New tumor markers are useful for early screening and diagnosis of prostate cancer, especially in patients with PSA gray areas. Diagnostic accuracy is improved by combining PSAD, fPSA%, and urine CXCL16.
Chemokine ligand16, which is highly expressed in prostate cancer tissues and blood, is a potential tumor marker. It was found that the combined detection of serum PSA and its parameters (fPSA, fPSA%, PSAD) and urine CXCL16 could improve the specificity, positive predictive value, negative predictive value and accuracy of the diagnosis of gray zone prostate cancer.
DD3PCA3:
DD3, now known as PCA3, is a prostate-specific gene discovered in 1999. It is located on chromosome 9q21~22 and expresses a non-coding mRNA with no protein product. PCA3 is closely related to prostate cancer (PCa), and its expression level in PCa tissues is significantly higher than that in normal prostate tissues and other disease tissues. Studies have shown that PCA3 is a specific marker gene for PCa. DD3PCA3 genes are strongly expressed in prostate cancer cells, are excreted from cancer cells, and are stably present in the blood, prostate massage, semen, and urine. In 2003, Hessels et al. showed that the DD3PCA3 levels of prostate cancer patients were much higher than those of non-cancer individuals, indicating that urine testing can effectively diagnose prostate cancer.
Fradet et al. used the uPM3 method to improve the accuracy of prostate cancer (PCa) diagnosis by detecting DD3PCA3 RNA and PSA mRNA in urine, with total sensitivity, specificity, positive and negative predictive values superior to serum tPSA. uPM3 analysis is particularly useful when serum PSA levels are low. The combination of uPM3 and tPSA can improve the detection rate and reduce tissue biopsy. However, accuracy relies on the proper operation of prostate massage. Improving DD3PCA3 RNA detection may further increase the detection rate of PCa, which has been approved by the FDA as a diagnostic marker for prostate cancer.
谷胱甘肽S转移酶P1(glutathione-S-transferase P1，GSTPl):
Aberrant methylation is closely related to tumor development. In normal cells, the gene regulatory region CpG island is unmethylated; After carcinogenesis, the CG region is methylated and can be used for tumor diagnosis. GSTPl gene mutations are associated with the development of PCa in humans, and the mutation leads to hypermethylation, which inactivates the gene and loses tumor suppressor activity. Measurement of GSTPl DNA methylation levels can improve the detection rate of early prostate cancer. Studies have shown that hypermethylated GSTPl can be detected in the urine of prostate cancer patients.
Goessl et al. pointed out that the detection of hypermethylated GSTP1 DNA in urine by methylation-specific PCR (MSP) can be used for prostate cancer diagnosis. Its specificity is 98% and its sensitivity is 73%, which is superior to serum PSA detection. There was no significant difference in the level of this indicator in the urine of patients at all stages. Gonzalgo et al. collected urine from patients after prostate puncture and used MSP to detect the level of supermethylated GSTP1 DNA. The results can divide patients into high- and low-risk groups and guide the need for biopsy monitoring. It's more concise and straightforward.
"Investigating biomarkers such as thymosin beta15 to improve prostate cancer diagnosis. Urine testing is easy to collect, non-invasive, or a new diagnostic pathway, but the technique needs to be simplified for clinical use. ”
4
Isolation and identification of exosomes in urine samples of patients with prostate cancer
Using high-throughput sequencing, Alicia Llorente's team at the University of Oslo Cancer Research Institute found that urine exosome-derived miRNAs can be used as biomarkers for monitoring patients at intermediate risk of prostate cancer, helping to distinguish tumor risk levels and treat them in a timely manner. The results were published in the British Journal of Cancer.
1
Isolation and identification of exosomes in urine samples of patients with prostate cancer
The authors extracted exosomes from the urine of 70 prostate cancer patients, detected the protein content by BCA and Qubit, and analyzed their characteristic proteins and particle size by Western blot and nanoflow cytometry. Steps:1. Exosome extraction: Exosomes are obtained by differential centrifugation from urine samples from prostate cancer patients. 2. Determination of protein content: BCA analysis and Qubit method were used to measure the total protein amount in exosomes. 3. Characteristic protein and particle size detection: Western blot technology was used to detect the characteristic protein of exosomes, and nano flow cytometry particle size analysis technology was used to evaluate its particle size.
Fig.1 Western blot identification of exosome-characteristic proteins
2
Exosomal differential miRNA identification
Next, the authors sequenced the isolated exosomal miRNAs and found that 5 miRNAs were the most abundantly expressed. These miRNAs were used to construct and validate the ISUP grading prediction model, and the results showed that these miRNAs were significantly different in the urine EVs of prostate cancer patients.
3
RT-qPCR validation analysis
The authors studied 60 patients with prostate cancer to verify differences in the expression of miR-186-5p, miR-320a-3p, and miR-30e-5p. Combined with serum PSA values, the model was constructed to improve the prediction accuracy of ISUP tumor classification.
Fig.2 miRNAs identified in urine EVs of prostate cancer patients showed differential expression patterns between ISUP grades
The authors identified miRNAs that distinguished ISUP grade 1-3 prostate cancer by miRNA sequencing, and verified the differential expression of miR-186-5p, miR-320a-3p, and miR-30e-5p in independent cohorts by RT-qPCR. Urine exosomal miRNAs show potential as biomarkers for prostate cancer. Combined with PSA detection, it can improve the reliability of risk level differentiation in intermediate-risk patients and provide a new method for active surveillance.
Prostate cancer in China is characterized by less incidence, more deaths, late staging of the first diagnosis of the disease, and increasing morbidity and mortality rates year by year [1], and the Lancet major prostate cancer report predicts that the number of prostate cancer cases worldwide will increase from 2.4 million in 2020 to 2.9 million in 2040 [2]. In this case, early screening, early diagnosis and early treatment of prostate cancer are particularly important for patients, and many technical means for early diagnosis of prostate cancer have been adopted in clinical practice to serve this purpose.
Figure 1: Overview of China's top 10 tumors in 2022[3]
Urine exosome prostate cancer detection products
"The Chinese Prostate Cancer Screening Guidelines 2022 prefer PSA screening, but it also faces limitations. In order to improve the accuracy, auxiliary diagnostic technologies such as "urine exosome prostate cancer detection" have been developed. Based on the FDA-approved EPI detection method, this technology establishes a risk model by detecting urine exosome information, and provides a diagnosis and treatment reference for people with PSA 4-20 ng/mL to reduce unnecessary biopsies. "
Figure 2: Urine exosomal RNA prostate cancer testing project process and applicable population
Exosomes are extracellular vesicles with a diameter of 30-100 nanometers secreted by a variety of cells and carry lipids, nucleic acids, proteins, etc. These vesicles are widely distributed in fluids such as urine and plasma, and their specific contents such as miRNA, mRNA, and lncRNA reflect the pathological state of blasts. Therefore, exosomes are considered as potential biomarkers for disease surveillance.
Figure 3: Biogenesis of exosomes [6]
Exosome enrichment
Automated, stable and efficient exosome enrichment and purification is a key link in scientific research and clinical translation, and it is also the basis for ensuring the accurate development of downstream experiments. To this end, Nukai Biotech has developed a fully automated exosome extraction system (EXODUS) and published the results as a cover article in Nature Methods [7]. The system combines a negative pressure oscillation system (NPO) combined with a dual-coupled harmonic oscillation system (HO) to act on the nanoultrafiltration chip, and the impurities such as free nucleic acids and proteins in the sample are quickly removed and retained through the nanopores, so as to purify and enrich exosomes. The advantages of this system are as follows:
High-speed secretion (up to 100 mL/h);
High purity and yield, heteroprotein removal efficiency > 99%, recovery rate > 90%;
Fewer samples, higher yields, supporting small, low-concentration, rare samples;
No exogenous impurities are introduced, and exosomes have complete morphology, high biological activity, and wide downstream applications.
Figure 4: Fully Automated Exosome Extraction System (EXODUS)
Launched the Urine Exosomal RNA Prostate Cancer Assay to provide risk assessment for 4-20 ng/mL serum PSA populations and reduce unnecessary punctures.
Figure 5: Differences in the outcomes of urine exosome prostate cancer detection and PSA screening methods for clinical decision-making
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