Biomarker Assays for Elevated Prostate-Specific Antigen Risk Analysis

Adil Abdulla; Stephen Leslie

Biomarker Assays for Elevated Prostate-Specific Antigen Risk Analysis

Author: Adil Abdulla Editor: Stephen W. Leslie Updated: 1/31/2026 4:59:30 PM

Introduction

Prostate cancer ranks among the most frequently detected malignancies in males, accounting for approximately 1.4 million new cases worldwide each year.[1] This disease represents the most commonly diagnosed cancer and the second leading cause of cancer-related mortality in men, with an estimated 2.4 million cases projected for 2040, according to the National Cancer Institute (NCI). Approximately 1 in 8 men develops prostate cancer over a lifetime. Despite this high incidence, the typically indolent disease course contributes to a mortality rate of only 1 in 41 diagnosed men.

One of the most effective approaches for screening, diagnosis, staging, assessment of therapeutic response, and prognostication in prostate cancer involves the use of serum or urine biomarkers for risk assessment. The NCI defines a biomarker as a biological molecule identified in blood, urine, other body fluids, or tissues that signals an abnormal process, condition, or disease. Appropriate application of biomarkers allows healthcare professionals to individualize diagnostic strategies while minimizing unnecessary diagnostic interventions and reducing the risk of overtreatment.

The Early Detection Research Network represents an NCI-led initiative focused on identifying, developing, and validating novel biomarkers and emerging technologies to improve the accuracy and timeliness of cancer diagnosis. These biomarkers may include proteins, DNA, messenger RNA (mRNA), metabolites, prostate cancer cells or their derivatives, exosomes, or measurements of cellular processes, eg, proliferation or apoptosis. Several commercial risk-stratification biomarkers now support clinical evaluation of patients with persistently elevated prostate-specific antigen (PSA) levels and suspected prostate cancer.

Beyond guiding clinical decision-making in low- or intermediate-risk patients with equivocal PSA values, surrogate biomarkers may be useful for assessing an individual patient’s response to a specific drug, procedure, or therapeutic intervention. Through this role, a biomarker can monitor treatment effectiveness for a defined disease or condition. Validated surrogate risk-stratification biomarkers frequently spare patients from prolonged clinical trials, unnecessary biopsies, costly imaging studies, or other invasive tissue-based diagnostic procedures.[2]

An ideal biomarker demonstrates high sensitivity and specificity, straightforward use and interpretation, cost-effectiveness, broad availability, reproducibility, and reliable quantification from an easily obtainable specimen. Additional essential characteristics include a negative predictive value of at least 90%, approval by the Food and Drug Administration (FDA), compliance with Clinical Laboratory Improvement Amendments (CLIA) standards, and endorsement by the National Comprehensive Cancer Network (NCCN).

Prostatic risk-stratification biomarkers serve primarily lower-risk and carefully selected borderline patients with mildly elevated PSA concentrations, generally ranging from 4 to 10 ng/mL, in whom unfavorable results often lead to avoidance of immediate additional testing, prostate imaging, biopsies, or other diagnostic procedures.[1][2] This activity examines the current landscape of available risk-stratification biomarkers as well as those under active investigation.

Etiology and Epidemiology

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Etiology and Epidemiology

The International Agency for Research on Cancer estimates that 1,414,259 new cases of prostate cancer are diagnosed worldwide yearly, resulting in 375,304 deaths. In the United States, the SEER (Surveillance, Epidemiology, and End Results) database estimates about 333,830 new cases and 36,320 deaths due to prostate cancer in 2026. Prostate cancer accounts for 15% of all new cancer diagnoses and 5.7% of cancer-related deaths annually. However, the 5-year relative survival is estimated at 98.6%.

The incidence of prostate malignancy has a geographical variation, with the highest in Australia/New Zealand, North America, and parts of Europe, primarily due to the employment of PSA testing and an aging population. Even though prostate cancer mortality has relatively less variation worldwide, the rates are exceptionally high among the Black population in the Caribbean and sub-Saharan Africa, intermediate in the United States, and lowest in South Central Asia.

Approximately 66% of prostate cancer cases are diagnosed in individuals older than 65, with the average age of initial diagnosis being 66. Although the exact etiology is unknown, genetic predisposition and family history are clearly associated with an increased risk of prostate cancer.[1][2] A small subset of patients with prostate cancer has a hereditary form of the disease. On average, these patients are diagnosed 6 to 7 years earlier than the general population. However, the clinical course and aggressiveness of cancers do not seem to differ.[3]

Genomic evaluation has identified more than 100 genes contributing to the risk of prostatic malignancy.[4][5][6] About 15% to 17% of the prostate cancer population has been identified as having germline mutations. Such mutations are independent of the cancer stage. A study of multigene testing among men with prostate cancer across the United States by Giri et al concluded that 15.6% of the study population had pathogenic variations of the genes studied, including BRCA1, BRCA2, HOXB13, MLH1, MSH2, PMS2, MSH6, EPCAM, ATM CHEK2, NBN, and TP53.[7][8] The most commonly found pathologic gene variation identified was BRCA2 (4.5%).[7] Nicolosi et al reported that 17.2% of the study population had a pathogenic germline variant, and genomic analysis could help identify high-risk families.[9][10][11]

The Identification of Men with a Genetic Predisposition to Prostate Cancer (IMPACT) study evaluated men aged 40 to 69 with BRCA1 or BRCA2 germline mutations by screening with PSA annually and targeted biopsy if PSA greater than 3.0 ng/mL.[12] After about 3 years of research, it was found that BRCA2 mutation carriers were more likely to develop prostate cancer, have a younger age of onset, and develop more clinically apparent tumors compared to noncarriers.[12]

Many exogenous factors are associated with the likelihood of developing prostate cancer, including metabolic syndrome, diabetes, obesity, dyslipidemia, Agent Orange exposure, and various dietary factors.[13] Japanese men have less risk of prostate cancer compared to Western men. However, the risk increases substantially if they migrate to the United States, implying an interplay of environmental, dietary, or lifestyle factors.[14] No proven dietary, lifestyle, or pharmacological therapy for the prevention of prostate cancer has been established, though.

Pathophysiology

Inflammation of the prostate gland, often caused by various genetic mutations, is the earliest sign of prostate cancer. This inflammation leads to oxidative damage, which causes telomeres at the ends of chromosomes to shorten, eventually initiating the development of prostate cancer. Several genes, including MYC, PTEN, NKX3.1, and the TMPRSS2-ERG gene fusion, have been identified in the initiation and development of prostate cancer, even though no single tumor suppressor gene is primarily responsible.

The TMPRSS2-ERG gene fusion activates the ERG oncogenic pathway, which is linked to disease emergence. The reactivation of cell cycle pathways leads to unchecked cell proliferation, promoting tumor metastasis. Gene expression profiling of metastatic disease identifies overexpression of EZH2 mRNA and proteins. Due to its role in apoptosis and proliferation, EZH2 is currently under research as a new prostate cancer target.

Specimen Requirements and Procedure

Current and investigational biomarkers, including blood-based and urine-based assays, improve diagnostic accuracy by supplementing PSA testing. These biomarkers include PSA derivatives (eg, PSA density and velocity) and newer molecular forms, eg, (-2) pro-PSA. Other significant biomarkers include the Prostate Health Index (PHI), the 4K score, prostate cancer antigen 3 (PCA3), and TMPRSS2-ERG gene fusions (see Table. Biomarkers Used for Prostate-Specific Antigen Testing).

Each biomarker offers unique advantages for detecting, diagnosing, and monitoring prostate cancer and is often integrated into risk assessment tools for better clinical decision-making. Their high negative predictive values allow clinicians to confidently rule out further testing in many cases, thereby preventing overdiagnosis and reducing the burden on patients and healthcare systems.

Table. Biomarkers Used for Prostate-Specific Antigen Testing

Biomarkers Tested	Commercially Available Tests	Specimen	Cutoff	Sensitivity	Specificity	Negative Predictive Value	Approval/ Certification	Recommendations by NCCN	At-Home Collection Kit
Total PSA Free PSA (-2) pro-PSA	Prostate health index (PHI)	Serum	27	90%	31.1% at sensitivity 90% [15]	89% for any prostate cancer 97% for clinically significant prostate cancer [16]	FDA	IB and RB	NA
Total PSA Free PSA Intact PSA hK2	4K Score	Serum	7.5%	89%	29%	95% [17]	FDA	IB and RB	NA
PCA3	PCA3	Urine	25	78%	57%	90% [18]	FDA	RB only	NA
Exosomal RNA (PCA3, RNA)	ExoDx Prostate Intelliscore	Urine	15.6	IB 92% RB 82%	NA	IB 91% RB 92% [19]	FDA	IB and RB	Available
PCA3 TMPRSS2-ERG gene fusion Serum PSA	My Prostate Score	Urine	No recommended cutoff	97%	33% [20]	90% for any prostate cancer [21][22]	FDA	IB and RB	NA
18 cancer-specific and high-grade cancer-specific genes	My Prostate Score 2.0	Urine, Serum	None	93%	44% [22]	95% [22]	CLIA	IB and RB (2B)	Available
HOXC6 DLX1 Serum PSA	Select MDx	Urine, Serum	PSA <10	89%	53%	95% [23]	CLIA	IB and RB	NA
Sarcosine Alanine Glycine Glutamate	Prostarix risk score	Urine	NA	NA	NA	NA	CLIA	NA	NA
Aqueous isoforms of PSA (structural variants)	IsoPSA	Serum	NA	90%-95%	47%	92% (89.3% to 94.8%)	FDA	IB and RB	NA
PSA, Free PSA, Free PSA%, MIC1, hK2, MSMB Genetic score from 254 SNPs HOXB13 SNP	Stockholm 3	Blood plus: Age, History, Prostate volume, and DRE	11%	92%-95 [24][25]	32.6%-40.7% [26]	83.6%	Lab Developed Test	IB	NA

IB, The clinical decision for the initial biopsy; RB, Repeat biopsy after prior negative biopsy; NA, Not Available.

The NCCN has recognized the PHI, 4K Score, ExoDx Intelliscore, My Prostate Score, My Prostate Score 2.0, Select MDx, and ConfirmMDx (a tissue confirmation test). Stockholm 3 is available as a Lab Developed Test (LDT).

Diagnostic Tests

Blood-Based Biomarkers

Prostate-specific antigen

PSA, a member of the kallikrein gene family, was first identified and isolated in 1979.[27] PSA is secreted by prostatic ductal and acinar epithelium and is highly organ-specific. PSA is circulated in free (fPSA) and protein-bound forms (complexed). The expression of PSA is androgen-dependent and is a crucial tool for the early detection, staging, treatment planning, and prognosis of prostate cancer. The widespread use of PSA testing in the 1990s led to increased prostate cancer diagnoses and a subsequent decline in mortality rates for this disease.

Although an excellent and sensitive biomarker, PSA is organ-specific rather than disease-specific. Prostatic basal layer and basement membrane violations allow PSA to escape prostate cells and enter the bloodstream, leading to an increase in serum PSA levels.[28][29] This increase in serum PSA levels may occur in several prostatic pathologies, eg, benign prostatic hyperplasia, prostatitis, or prostate malignancy, or transiently with prostatic manipulation, as in prostate biopsies, prostatic massage, or urethral catheterization.[27][30][31][32][33]

Treatment with 5-alpha-reductase inhibitors has been shown to reduce PSA levels by approximately 50% when treatment is continued for 6 or more months.[34][35] Despite this, multiple guidelines still recommend PSA as the primary tool for initial prostate cancer screening. However, experts have yet to agree on the subset of patients to screen, the appropriate age to start screening, and other relevant factors to consider. All guidelines recommend that prostate cancer screening be an informed decision made after shared decision-making and a risk-benefit analysis. The most scientifically correct and reasonable guideline is from the American Urological Association (AUA), as it takes into account high-risk factors, the known incidence of prostate cancer appearing in some individuals before the age of 50, and the longer life expectancy of men, which now averages 87 years.[13]

The following recommendations have been established by the AUA and the Society for Urologic Oncology (SUO):

PSA screening in men younger than 40 is not recommended.
Screening may be offered to men at high risk starting at age 40, eg, those of Black ethnicity, those with a family history of prostate cancer, and those with BRCA1 and BRCA2 germline mutations.
Routine screening for men at average risk may be offered starting at age 45.
Two separate high PSA levels are necessary before further investigations are performed.
Shared decision-making is recommended for men when PSA screening is being considered, with a decision based on the individual's values and preferences.
Regular screening may be offered every 2 to 4 years, but can be personalized based on patient preferences, age, PSA, prostate cancer risk, comorbidities, life expectancy, and general health considerations.
Men with a reasonable life expectancy of 10 years or less should not be offered routine scre,ening as they are unlikely to benefit from it.
Routine PSA screening in men aged 70 and older should be based on shared decision-making and individual patient preferences, life expectancy, and general health.
Age-adjusted PSA normal values may be used, particularly in patients older than 70, where the upper normal total PSA level is 6.5 ng/mL (5.5 ng/mL for African Americans and other high-risk groups).[27][36][37]
Screenings may be discontinued or the interval substantially lengthened for patients aged 75 or older if the PSA is <3 ng/mL.[13][38][39]

A baseline PSA level that predicts the likelihood of prostate malignancy detection and the chance of developing high-grade prostate malignancy can customize the rescreening intervals. According to several studies, screening intervals of 2 to 4 years are unlikely to miss curable prostate cancer.[38][40][41] Therefore, the AUA recommends a 2-year screening as a reasonable approach. The AUA suggests that men older than 60 with PSA levels below 1.0 ng/mL may benefit from prolonged screening intervals, eg, 2 to 4 years.[38]

Prostate risk stratification biomarkers

The primary purpose of prostate risk stratification biomarker tests is to identify patients with a negative result who do not require further immediate testing, imaging, or biopsies.[13] These biomarkers are primarily intended for use in lower-risk and selected borderline patients, a subset with PIRADS 3 lesions on multiparametric magnetic resonance imaging (MRI), with slightly elevated levels of PSA, typically 4 to 10 ng/mL. In these cases, a negative test result can reasonably halt additional testing, prostate imaging, biopsies, and other diagnostic procedures, allowing for the resumption of routine screening.[1][13] Because of their high negative predictive value, these tests help clinicians determine that, given the low likelihood of detecting a clinically significant malignancy with further evaluation, immediate investigation is unnecessary.[1]

However, this approach is not recommended if the total PSA is >10 ng/mL or the patient is otherwise in the high-risk category. The high-risk group includes men of African descent; those with a family history of prostate malignancy; those with germline mutations that increase the risk for prostate cancer, eg, BRCA1, ATM, CHEK2, BRCA2; men with a family history of multiple malignancies or Lynch syndrome; and those with known exposure to Agent Orange.[13][42][43][44][45][46] These patients should proceed with a biopsy, as a clinically significant malignancy is more likely than average. Therefore, clinicians can generally skip prostatic risk-stratification biomarkers in high-risk patients with elevated PSA levels.[1]

Prostate-specific antigen derivatives

Clinical decision-making is enhanced by using PSA derivatives, eg, PSA density, velocity, doubling time, age-adjusted values, and, recently, different molecular derivatives. PSA velocity is the rate of change of PSA over a year, whereas PSA density is the PSA-derived computation of total PSA (tPSA) divided by prostatic volume. The PSA doubling time measures how quickly a rising PSA level doubles, gauging the rate-dependent exponential rise in serum PSA over time. These PSA-derived metrics are used for decision-making regarding prostate biopsy in an early detection setting.

Among the standard derivatives, PSA density provides substantially more predictive information than PSA velocity. A higher PSA density has a greater likelihood of clinically significant prostate cancer, particularly in smaller prostates, when a value of 0.15 ng/mL/cc was considered.[47]

Research suggests that a PSA density of 0.1 to 0.15 ng/mL/cc or higher is considered significant (higher risk). In contrast, a value lower than 0.09 ng/mL/cc is considered insignificant (<4% chance).[48][49][50] A PSA density of 0.15 ng/cc is typically the cutoff point.[51][52][53][54] PSA velocity and doubling time have limited diagnostic value but may aid in prognostication.[55]

Studies have shown a correlation between a low percentage of fPSA (free PSA) and aggressive pathological features.[56][57] The percentage of fPSA production was lower in malignant prostates compared to those of men without.[58][59][60][61] This observation led to the development of fPSA testing as an adjunct to improve the accuracy of total PSA as a prostate cancer biomarker.[62][63]

The United States FDA approved the use of fPSA as a biomarker to enhance screening in males with blood PSA levels of 4 to 10 ng/mL and a negative digital rectal examination. A percentage fPSA cutoff of 18% has improved the detection of prostate cancer in men when compared to total PSA (tPSA) alone. Such patients are recommended for prostate biopsy. The prostate malignancy detection was 56% in men with a percentage of fPSA less than 10 %, compared to 8% when the percentage of fPSA was greater than 25%.[63][64]

The NCCN recommends incorporating the percentage of fPSA for decision-making for prostate biopsy and recommends a cutoff of 10%.[65] However, it has no clinical implication in patients with tPSA greater than 10 ng/mL or in the follow-up of known prostate cancer patients. The use of predictive calculators incorporating multiple clinical variables, eg, tPSA, fPSA percentage, and digital rectal examination (DRE) findings, for shared decision-making has increased.[66][67][68]

Free prostate-specific antigen isoforms

The serum isoforms of fPSA, namely pro-PSA, benign PSA, and intact fPSA, exist in equal concentrations in serum and are promising biomarkers. Studies have shown significant levels of truncated forms of PSA in malignant prostate tissues, eg, (-2) pro-PSA. (-2) pro-PSA has been validated as a screening biomarker before a prostate biopsy and is also a strong predictor of histological aggressiveness, as indicated by a Gleason score of 7 or higher.[15][16]

Studies have shown that (-2) pro-PSA is superior to PSA and fPSA percentage as a biomarker in predicting prostate malignancy in men planned for biopsy with a serum PSA of 4 to 10 ng/mL. In addition, it correlates with the tumor's pathological aggressiveness.

Human kallikrein 2 and the 4K score

Human kallikrein 2 (hK2) shares many similarities with PSA. They are both regulated by androgens, are proteases, and have an identical specificity for the prostate.[69] However, in contrast to PSA, hK2 is a much more potent protease and is selectively expressed in cancerous tissue, where its expression correlates with the neoplasm/s aggressiveness, degree of differentiation, and risk of biochemical recurrence.[69] Combining fPSA with hK2 has increased the cancer detection rate in the 4 to 10 ng/mL PSA range.

In the 4K score assay, the 4 kallikrein proteins—tPSA, fPSA, intact PSA, and hK2—are integrated. The 4K score predicts the likelihood of biopsy-positive prostate cancer when paired with clinical characteristics, including age and prior negative biopsies. This biomarker also accurately predicts cancer aggressiveness.[70] Meta-analysis has shown that the 4K test improves the predictive accuracy of biopsy by 8% to 10% and reduces the rate of unnecessary biopsies by 48% to 56%.[71] The 4K score also predicts the risk of distant metastasis even 20 years later in men with a PSA of 2 ng/mL or greater. The 4K score has been included in the NCCN prostate cancer early detection guidelines.[72]

IsoPSA

The IsoPSA blood test examines structural variations in the PSA protein associated with high-grade malignancy. It uses a 2-phase aqueous collection system to separate and identify PSA structural isoforms that are associated with malignancy.[73][74] In practice, it is clinically similar to the 4K, PHI blood tests, and the ExoDx urine test, although no head-to-head studies have been performed. IsoPSA is FDA-approved, recommended by the NCCN, and included in their prostate cancer early detection guidelines.

Prostate health index

PHI is a blood-based biomarker that can predict the risk of aggressive prostate malignancy, indicated by a Gleason score of 7 or higher, at biopsy. This assay analyzes tPSA, fPSA, and (-2) pro-PSA levels. PHI was approved in 2012 by the FDA to diagnose prostate cancer in men aged 50 and older with a clinically negative DRE and PSA of 4 to 10 ng/mL.[75][76]

PHI has consistently shown superiority over tPSA and fPSA in the early detection of prostate cancer. As a result, it has been added to various web-based risk assessment calculators, eg, the Prostate Cancer Prevention Trial (PCPT) calculator.[15][77] In 2015, the NCCN recommended using PHI for early cancer detection. However, due to limited studies in the United States population, PHI is not recommended as first-line screening for all patients.

Serum protein panel

The 3 prostate cancer biomarkers—FilamenA, FilamenB, and Keratin19—have been combined into a novel serum protein panel for disease screening and prognosis, based on recent studies. A panel of these biomarkers with PSA outperformed PSA alone in diagnosing, predicting aggressiveness, and differentiating cancer from benign prostatic hyperplasia.[78]

Stockholm3

Stockholm3 incorporates a combination of clinical history, DRE findings, prostate volume, and age, together with multiple blood tests (PSA, Free PSA, Free PSA%, MIC1, hK2, MSMB, a genetic score (from 254 SNPs), and HOXB13.[24][25][26] Developed in Sweden, Stockholm3 has already been used in over 90,000 men and is now available in the US as a Lab Developed Test. This testing modality has a sensitivity and specificity similar to other approved prostate cancer biomarkers and is recommended by the NCCN as an aid to help identify higher-risk patients who can safely avoid an initial biopsy.

Urine-Based Biomarkers

Annexin A3

Annexin A3, a calcium-dependent phospholipid-binding protein, is found to be negatively associated with the detection of prostate malignancy when present in urine. Researchers have evaluated the utility of analyzing Annexin A3 levels in post-DRE urine as a stand-alone or in conjunction with serum PSA for clinical decision-making before a prostate needle biopsy.[79]

Investigators showed that Annexin A3 enhanced PSA's ability to predict malignancy in needle biopsy with an area under the curve (AUC) of 0.81. Cao et al studied a multiplex assay combining urine annexin A3, PCA3, TMPRSS2: ERG, and sarcosine for prostate biopsy decision-making.[80] The multi-marker assay was accurate, with an AUC of 0.84 in patients with a PSA of 4 to 10 ng/mL.[80]

ExoDx prostate intelliscore

Exosomes are tiny extracellular vesicles (40-150 nm) released by various cell types, including cancerous cells. They contain lipids, proteins, and nucleic acids. Prostate-derived exosomes serve as diagnostic and prognostic biomarkers as they are a source of proteins and microRNA.[81][82] Urinary exosome analysis offers benefits, including the stability of exosomes owing to their lipid bilayer, which protects their genetic content from enzymatic degradation. No unpleasant prostatic massage is needed before collecting the sample, and the test can be performed at home with the specimen mailed directly to the laboratory by the patient, saving staff time and paperwork. Exosomal RNA and protein content may be superior to less stable, chemical, nongenetic-based biomarkers.

ExoDx prostate intelliscore is a urine exosomal RNA-based assay to assess the expression of the prooncogenic genes ERG, PCA3, and SPDEF and generate a score. The test is recommended for males older than 50 with a PSA of 2 to 10 ng/mL who are being evaluated for a preliminary biopsy. This test is used with other standards of care criteria to assess the likelihood of Gleason 7 and above prostate cancer on an initial biopsy.[83][84]

This assay helps safely reduce unnecessary biopsies. This score predicted high-grade cancer (Gleason 7 or higher) with a negative predictive value of greater than 90%.[83] The test is far more patient-friendly compared to similar bioassays, as it does not require a digital rectal exam or prostatic massage, unlike previous urine-based tests for prostate cancer. The kit is designed for home use and can be mailed directly to the diagnostic laboratory by the patient, making it particularly useful for patients without a rectum for digital prostatic massage or those for whom the procedure is not feasible.

A large cohort study involving 774 patients in the United States found that the ExoDx assay, in conjunction with standards-of-care features, outperformed the ExoDx assay or the standards-of-care variables when used alone in predicting the presence of Gleason grade 7 or higher and negative biopsies.[83] Several exosomal metabolites, including survivin and claudin 3, have also been higher in the plasma of patients with prostate cancer.[85][86][87] ExoDx is also among the tests recommended by the NCCN for prostate cancer risk-stratification bioassay testing.

Prostarix risk score

This urine-based assay helps clinicians decide whether to biopsy in the grey zone, eg, when DRE is negative, patients are undecided, or PSA is only mildly elevated.[88] The assay quantitatively analyzes 4-metabolites, sarcosine, alanine, glycine, and glutamate, in urine employing liquid chromatography-mass spectrometry. The quantitative analysis of these amino acids helps to calculate the risk score and detect malignancy-related metabolic abnormalities. Studies have shown that the panel's performance in diagnosing prostate cancer (AUC=0.64) was superior to that of serum PSA (AUC=0.53) and the PCPT calculator alone (AUC=0.61).[89]

Prostate cancer antigen 3

The ease of collecting urine specimens and the knowledge of prostate cell exfoliation in urine have made urine a source for potential biomarker research.[90] The transcriptome comparison of prostate cancer and normal tissues led to the identification of PCA3.[91] Formerly known as differential display code 3, PCA3 is a long noncoding mRNA. PCA3 does not encode a protein, but its mRNA transcripts can be detected and measured in urine from prostatic cytology.[92] The PCA3 gene is overexpressed in 95% of primary prostate cancer specimens but not in benign prostate tissue.[93][94]

Reverse transcription polymerase chain reaction (RT-PCR) evaluation revealed that PCA3 performs better compared to PSA in identifying prostatic malignancy.[95][96] Recently, with the advent of a transcription-mediated amplification assay, sensitivity has improved compared to RT-PCR.[97]

The commercial PCA3 assay was FDA-approved in the United States in 2012 to assist decision-making in men aged 50 and older with a prior negative biopsy. The European Association of Urology also recommends this test. A urine sample for PCA3 testing is obtained immediately after a prostate massage or DRE. Substantial research has examined the diagnostic value of PCA3 in post-prostatic massage urine, and all studies have shown that PCA3 scores closely correlate with the likelihood of a positive biopsy.[98][99][100] Unlike PSA, PCA3 levels are independent of prostate size.[101]

Studies have reported a 14% positive biopsy rate when PCA3 levels are less than 5, compared to 70% when PCA3 is greater than 100.[99] PCA3 has been shown to exhibit varying sensitivity, specificity, and predictive values across alternative cutoffs. The FDA used a threshold of 25 for its approval. Studies involving large patient cohorts have demonstrated the superiority of PCA3 over PSA alone for diagnosing prostate cancer.[102][103][104] PCA3 has been incorporated with other clinical parameters into various nomograms, eg, the web-based PCPT risk calculator, to assess the likelihood of a positive biopsy.[105]

Prostate cancer cell lines in urine

Prostate cancer cells can be detected in post-DRE urine using multiplex immunofluorescence cytology staining for AMACR, Nucleolin, DAPI, and Nkx3.1. In a study involving a cohort of 50 patients, this method achieved a sensitivity of 36% and a specificity of 100%.[106] Immunohistochemistry also studies cell lines for ERG, AMACR, and Prostein (prostate epithelium-specific).[107] In a cohort of 63 individuals, this assay has a sensitivity of 64% and a specificity of 68.8% for diagnosing prostate cancer.[107] The NCCN has recognized the PHI, 4K score, ExoDx intelliscore, My Prostate Score, Select MDx, and ConfirmMDx (a tissue-based confirmation test).

Select MDx

The Select MDx is an RT-PCR assay that analyzes urine specimens after DRE, along with other chemical risk factors, to facilitate early detection of prostate malignancy and decision-making regarding biopsy. This 3-gene panel assay has enhanced the identification of high-grade cancer by integrating HOXC6, TDRD1, and DLX1 mRNA levels in urine with other clinical indicators. In addition, Van Neste et al have validated it against various clinical decision-making tools, including the PCA3 assay and the PCPT risk calculator.[108] The NCCN has identified Select MDx as one of its recommended prostate cancer risk stratification assays.

TMPRSS2-ERG gene fusions and My Prostate Score 2

With the discovery of gene fusions in prostate malignancy, considerable research has been performed to harness their utility as prostate cancer biomarkers. The most common gene alteration in prostate cancer is the fusion of the androgen-regulated gene transmembrane protease, serine 2 (TMPRSS2), with the N-terminal-deleted ERG coding region.[109] Between 50% and 60% of patients with prostate cancer of European ancestry have this gene fusion, which results in the oncogenic activation of ERG.[110] The use of gene fusion was most significant when combined with other biomarkers, eg, PCA3.

A multicentric study involving 1312 men compared post-DRE TMPRSS2:ERG/PCA3 ratios with serum PSA. The study demonstrated the clear superiority of the TMPRSS2:ERG panel with PCA3 in detecting clinically significant malignancy at biopsy.[111] An assay developed by the University of Michigan, My Prostate Score 2 (MPS2), combines serum PSA, urine PCA3, and urine TMPRSS2:ERG gene fusion to estimate the probability of identifying prostate malignancy on biopsy and the likelihood of aggressive disease.[112] PCA3 and TMPRSS2:ERG gene fusion is presently incorporated into a PCPT risk calculator for decision-making for prostate biopsy in patients with persistently (at least 2) elevated levels of PSA to avoid unnecessary biopsies safely.[105] The NCCN has included MPS2 among its recommended prostate cancer risk-stratification bioassays.

Recommended Use of Risk-Stratification Bioassays in Prostate Cancer Screening

Although their precise role in prostate cancer screening is still being determined, experts have suggested that risk-stratification bioassays are most helpful in patients with persistently elevated PSA levels who are low-risk, borderline, or equivocal cases. It may also be used in high-risk patients who wish to avoid a biopsy if possible or are at increased risk of biopsy-related complications. In such cases, the result, if negative, is used to help make a clinical decision not to pursue further prostate cancer diagnostic testing or screening procedures immediately.[1][13]

Regardless of the specific test used, about 75% will be positive. This result is not an indication of cancer, only that the risk analysis biomarker assay could not safely exclude a possible clinically significant cancer. The majority of these patients ultimately demonstrate negative biopsies and do not have prostate cancer.

Risk stratification bioassays for prostate cancer may also have a role in monitoring patients on active surveillance and those with high-grade prostatic intraepithelial neoplasia or atypical small acinar proliferation, which are considered premalignant.[1][113][114][115][116][117] The risk calculators can be accessed from the following websites:

Prostate Cancer Prevention Trial Risk Calculator 2.0 (UT Health San Antonio)
The Prostate Cancer Risk Calculators (SWOP—The Prostate Cancer Research Foundation, Reeuwijk)
Prostate Cancer Prevention Risk Calculator (UNC School of Medicine)
SPCC Risk Calculator (Stanford Medicine)

Testing Procedures

An abnormally elevated PSA in a grey zone (4-10 ng/mL) warrants a repeat PSA and a DRE. Patients whose repeat PSA and DRE are normal may continue with routine follow-ups. However, if the repeat PSA is elevated or the DRE is abnormal, and a prostate malignancy is suspected, biomarkers aid in the decision-making regarding continuing observation versus proceeding directly to a biopsy. Various biomarkers, including PHI, 4K score, PCA3, ExoDx Intelliscore, and Select MDx, help make this decision by identifying patients at low risk.

Biomarkers may be used alone or as a variable in risk analysis calculators. For example, if a biopsy has already been performed with a negative result, biomarkers can help determine when, or whether, to repeat the biopsy. According to the NCCN recommendations, if the initial biopsy is reported as atypia, suspicious for cancer, or high-grade prostatic intraepithelial neoplasia, biomarkers that improve the specificity of screening, including fPSA, 4K score, PHI, PCA3, Exo Dx Prostate score, and MPS2, may be considered (see Table. Urinary Biomarkers). Alternatively, this subset of patients may proceed with a multiparametric prostate MRI if no prior prostatic study is available.

Most urinary biomarker assays are performed after prostatic manipulation (massage), except the ExoDx EPI score, which is not required. Studies have shown that a DRE can enrich urinary biomarkers and enhance yield; the first void after a DRE is obtained and analyzed. Following a prostate massage, the first 10 to 50 mL of urine voided is centrifuged at 1,000 to 2,000 rotations per minute to produce urine precipitates that may contain prostate cancer cells, cell fragments, and various biomolecules, eg, DNA, RNA, and proteins. Free proteins, DNA, RNA, other tiny molecules, and exosomes are all detected in the supernatant.

Exosome precipitation occurs as a result of further ultracentrifugation of the supernatant. The ExoDx Intelliscore is far more patient-friendly compared to other biomarkers, as it does not require a digital rectal exam or prostatic massage. Unlike other urine-based tests for prostate cancer, the ExoDx Intelliscore is available as a kit for home usage that patients can mail directly back to the diagnostic laboratory.[92]

Table. Urinary Biomarkers

Assay Name	Requires Prostatic Manipulation/Digital Rectal Exam	Specimen Used
PCA3	Post-DRE void	Whole urine/precipitate
My Prostate Score 2.0 (PCA3, TMPRSS2-ERG)	Post-DRE void	Whole urine
Annexin A3	Post-DRE void	Precipitate
ExoDx Intelliscore	Not necessary/not recommended	Precipitate
DLX1, HOXC6	Post-DRE void	Precipitate
Prostate cancer cell lines	Post-DRE void	Precipitate

Clinical Significance

Serum PSA is the gold standard during initial investigation for initial prostate cancer screening and early detection. PSA as a biomarker is a sensitive tool, but its specificity is low. Various benign conditions, eg, benign prostatic hyperplasia and prostatitis, and manipulations, eg, DRE, biopsy, and catheterization, can increase PSA levels. Consequently, a diagnosis with PSA alone can lead to overdiagnosis and unwanted biopsies, adding pressure on healthcare systems and causing unnecessary patient anxiety.

A PSA level eliminating the risk of prostate cancer does not exist. Instead, the likelihood of prostate cancer and an aggressive disease increases as the PSA rises. Instead of using an absolute PSA level to establish if a prostate biopsy is necessary, the clinician should consider other variables, including prostate volume, inflammation, medications, patient age, life expectancy, prior PSA levels, and comorbidities.

Prostate cancer biomarkers assist the clinician in decision-making in patients with PSA levels in the grey zone of 4 to 10 ng/mL to safely avoid unnecessary biopsies. The biomarkers can also predict clinically significant tumors and help tailor the treatment regimen. Biomarker assays are employed in a grey zone PSA level setting with a negative DRE. Biomarkers allow clinicians to discuss better-informed decision-making with patients.

Various additional tools, eg, PSA derivatives, PSA kinetics, PSA molecular forms, PSA density calculations, and risk-stratification biomarkers, can be used as standalone or in multiplex panels to arrive at an informed decision in equivocal situations. The new prostate cancer risk-stratification biomarkers are intended to assist clinicians in educating patients and facilitating optimal shared decision-making.

Enhancing Healthcare Team Outcomes

Optimal use of risk-stratification biomarker assays for prostate cancer can significantly reduce unnecessary diagnostic procedures and treatments. The healthcare team should be familiar with implementing these assays when appropriate and help educate the patient and family when necessary or requested. The screening, diagnosis, and treatment must be individualized based on age, high-risk characteristics, comorbid conditions, life expectancy, and personal preferences. The patient must be empowered to make appropriate decisions through interdisciplinary team consultations and effective communication between the patient and clinicians. Overtreatment, due to adverse effects, can cause harm to the patient, adversely affecting his quality of life and placing the patient under undue psychological distress. Eligible patients may be spared from such adversities by utilizing active surveillance protocols.

The past decade saw a paradigm shift in the management of prostate malignancy. With advances in diagnostics, imaging, genetics, and metabolomics, the evaluation and management of prostate malignancy have become even more complex. However, there needs to be more consensus on various diagnostic and treatment protocols, and different expert guidelines fall short of offering concrete recommendations. Interprofessional agreement and coordination are crucial for improving prostate cancer outcomes.

To provide the patient with the best care possible, a team of specially qualified nurses, technicians, general practitioners, advanced practitioners, pharmacists, genetic counselors, psychotherapists, oncologists, radiologists, urologists, and allied healthcare workers must collaborate. Physicians and general practitioners evaluate risk, interpret biomarker results, and integrate findings into shared decision-making. Advanced practitioners support longitudinal monitoring and patient counseling. Nurses reinforce education, ensure adherence to testing and follow-up, and monitor adverse outcomes. Pharmacists contribute by assessing therapeutic response and medication safety, while other health professionals facilitate diagnostic coordination and patient navigation. Effective communication and collaboration across disciplines enhance patient-centered care, improve outcomes, strengthen safety, and optimize team performance.

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