Clinical Biofluid Assays for Prostate Cancer

Clinical Biofluid Assays for Prostate Cancer: Comparison

Please note this is a comparison between Version 2 by Peter Tang and Version 1 by Gyorgy Petrovics.

Prostate cancer (PCa) is a heterogeneous disease, with a large percentage of prostate tumors being indolent, and with a relatively slow metastatic potential. However, due to the high case numbers, the absolute number of PCa-related deaths is still high. In fact, it causes the second highest number of cancer deaths in American men. As a first step for the diagnosis of PCa, the PSA test has been widely used. However, it has low specificity, which results in a high number of false positives leading to overdiagnosis and overtreatment. Newer derivatives of the original PSA test, including the Food and Drug Administration (FDA)-approved 4K (four kallikreins) and the PHI (Prostate Health Index) blood tests, have higher specificities. Tissue-based PCa tests are problematic as biopsies are invasive and have limited accuracy due to prostate tumor heterogeneity. Liquid biopsies offer a minimally or non-invasive choice for the patients, while providing a more representative reflection of the spatial heterogeneity in the prostate. In addition to the abovementioned blood-based tests, urine is a promising source of PCa biomarkers, offering a supplementary avenue for early detection and improved tumor classification. Four urine-based PCa tests are either FDA- or CLIA-approved: PCA3 (PROGENSA), ExoDX Prostate Intelliscore, MiPS, and SelectMDx.

prostate cancer
liquid biopsy
blood-based and urine-based biomarkers

1. Introduction

PCa is the second most prevalent cancer, with nearly 1.4 million new cases and 375,000 deaths globally, making it the fifth leading cause of cancer death among men in 2020 [1,2]^[1][2]. Specifically in the United States, PCa incidence for 2023 was predicted to be 288,300 new cases and 34,700 deaths ^[3]. When evaluated across 36 nations, countries in Africa reported the highest mortality associated with PCa ^[4]. It was noted that the incidence of this disease is markedly higher in African and African American populations, often manifesting in a more aggressive and lethal form. One hypothesis attributing to these racial disparities is the presence of genomic variations. Germline susceptibilities specific to various races have been shown through genome-wide association studies, suggesting differential genomic architecture amongst diverse racial groups [5,6,7]^[5][6][7]. For instance, African men display a genetic risk score approximately 2.18 times higher than European men, whereas East Asian men exhibit a risk 0.73 times lower than European men ^[8].

Remarkably, PCa differs from many other malignancies because a large fraction of prostate tumors is indolent with a relatively low metastatic potential. Consequently, many patients may remain asymptomatic and free from complications for prolonged periods of time. Even in cases with metastatic spread, effective management can extend the survival rate. However, if the cancer cannot be controlled, it will progress rapidly, leading to multiple adverse symptoms and death. Therefore, monitoring the status of developing PCa is essential for its treatment.

Early detection strategies for PCa have been prominently dependent on the PSA blood test for decades. Another commonly used screening method for PCa detection is digital rectal examination (DRE), usually performed after an elevated serum PSA level is detected. However, neither the PSA test nor DRE provide enough information to diagnose PCa or to assess its risk of becoming an aggressive cancer. Abnormal results of these tests, however, warrant diagnostic prostate biopsies for pathological examination and, if positive, cancer staging. Cancer-positive diagnostic biopsies are still the only accepted way to diagnose PCa.

A brief introduction of the more recent biofluid-based clinical tests will be reviewed here (Table 1 and Table 2). Two additional blood tests were introduced to clinical use that are better than PSA alone. The PHI test measures three PSA forms in patients with an initial serum PSA of 4–10 ng/mL (“gray zone”). It performs well in predicting the presence of PCa (AUC of 0.7) and similarly well for the presence of high-grade PCA (GS ≥ 7). The four-kallikrein (4K) score, measuring four kallikreins, combined with clinical information, is used to estimate the likelihood of high-grade PCa on biopsy (AUC of about 0.8).

Table 1.

Current FDA- or CLIA-approved biofluid-based biomarkers in prostate cancer.

Biomarker Test	Molecular Markers

Biomarker Test		Approval
	Urine Sample	Advantages	Limitations
Serum-based
Progensa PSA3 Assay, Hologic, Marlborough, MA, USA	Post-DRE	Assay score correlates with the likelihood of repeat biopsy	Sample collection requirements, clinical data and risk factors should be considered before decision, certain conditions, procedures and medications may influence assay score
Prostate Serum Antigen
ExoDX (Prostate Intelliscore), Exosome Diagnostics Inc., Waltham, MA, USA	PSA	FDA
	Non-DRE	Non-invasive test, at-home collection kit, assay score correlates with risk for advanced PCa	All clinical data should be considered before decision	Prostate Health Index, PHI, Beckman Coulter Inc., Brea, CA, USA	Total PSA, fPSA, p2PSA

ExoDX Prostate (Intelliscore), Exosome Diagnostics Inc., Waltham, MA, USA
	Exosomal RNA (PCA3, ERG)	CLIA
MiPS, MyProstateScore, MCTP, Arbor, MI, USA
MiPS, MyProstateScore, MCTP, Arbor, MI, USA	FDA
	Post-DRE	Assay score correlates with a need for biopsy	All clinical data required to generate final score and should be considered before decision	4Kscore, OPKO Health company, BioReference Laboratories Inc., Township, NJ, USA
SelectMDX, MDx Health, Irvine, CA, USA	Total PSA, fPSA, intact PSA, hK2	Post-DRE CLIA
		Assay score predicts advanced PCa in biopsy	Sample collection requirements, all clinical risk factors should be considered before decision	Urine-based
Progensa PSA3 Assay, Hologic, MA, USA	PCA3	FDA
PCA3	, TMPRSS2-ERG	CLIA
SelectMDX, MDx Health, Irvine, CA, USA	HOXC6, DLX1	CLIA

Table 2.

Urine-based assays: advantages and limitations.

2. PSA-Related Blood-Based Assays

2.1. Serum PSA

PSA, a protein found in blood, is produced by both normal and malignant prostate epithelial cells within the prostate gland. PSA is a 34-kDa glycoprotein comprising 240 amino acids, exclusively produced by prostatic epithelial cells. As a serine protease from the kallikrein gene family, it shares similarities with human glandular kallikrein and exhibits activities like chymotrypsin, trypsin, and esterase. Predominantly in serum, PSA binds with alpha 1-antichymotrypsin, and its production seems to be regulated by circulating androgens acting through the androgen receptors. The role of serum PSA as PCa biomarker was first discovered in 1979 [9,10,11]^[9][10][11].

The PSA test determines the amount of PSA in nanograms per milliliter (ng/mL) of blood. Historically, a PSA reading below 4 ng/mL is considered normal, while higher levels warrant an additional PSA test and DRE. The Food and Drug Administration (FDA) first endorsed the PSA test in 1986 to track prostate cancer in already diagnosed patients. In 1994, the FDA approved the PSA test to be used in conjunction with DRE to aid in the detection of prostate cancer in men. Periodic PSA screening for men between 55 and 69 years old is suggested but should be individualized, with consideration for potential benefits and harms, due to the low specificity of the serum PSA test. Aside from PCa, benign conditions like prostatitis (inflammation of the prostate) and benign prostatic hyperplasia (BPH) (an enlarged prostate) can also elevate PSA levels. In addition, PSA levels can also change due to age, prostate size, trauma, lifestyle, and certain medications. Even though the PSA test has its limitations, its utility in early disease detection and post-treatment monitoring remains valuable.

2.2. Prostate Health Index (PHI)

While the screening of total PSA remains contentious due to its limited specificity in detecting clinically significant PCa, advances in biomarker research have attempted to refine this diagnostic approach. The low specificity of the PSA test may result in unnecessary biopsies leading to the detection of no tumors, or indolent tumors that would not have caused harm during the patient’s lifetime. Given the limitations of the PSA test, a composite metric called the Prostate Health Index (PHI) was proposed. PHI combines the total and free PSA with [−2] proPSA (p2PSA), using the formula p2PSA/(free PSA) × √PSA as a “PHI score”. PSA in the blood is usually bound to other proteins but tends to circulate as “free” PSA in the case of benign prostatic hyperplasia (BPH) or PCa. A ratio of free PSA to total PSA level is used for prostate disease assessment. PSA precursors were found to be a more specific serum marker for PCa. In a study involving 1091 serum samples from patients subjected to prostate biopsies, these precursors emerged as potent predictors of PCa, especially in the PSA range of 2 to 4 ng/mL ^[12].

One precursor, the [−2] proenzyme PSA, displayed an ability as a predictor of PCa in a cohort of 123 men scheduled to undergo prostate biopsies ^[13]. The use of this precursor and the free PSA to total PSA ratio enhances the interpretative power of elevated PSA levels, increasing the likelihood of identifying PCa through biopsies. Specifically, the PHI score is suggested for men aged 50 or above, with total PSA results between 4 and 10 ng/mL and negative DRE findings.

PHI was approved by the FDA and is now commercially available in regions including the USA, Europe, and Australia. Notably, in a multicenter prospective trial involving 658 men, PHI displayed superior diagnostic capabilities compared to its individual components, with an AUC of 0.708 ^[14]. Moreover, another study with 506 participants underscored PHI’s ability to considerably reduce biopsy procedures (36.4% vs. 60.3%) in the control group ^[15]. Additionally, in a study of >700 patients, while comparing PHI score and other metrics like BPH-associated free PSA, tPSA, fPSA, and p2PSA, PHI exhibited significant increases in PCa predictive value and specificity of PHI ^[16].

A comprehensive meta-analysis of sixty studies, including 14,255 individuals, illustrated PHI’s better PCa diagnostic efficacy over PSA. Pooled results unveiled that PHI had sensitivities of 0.791 and aspecificity of 0.625 for PCa detection, with clinically significant PCa detection sensitivity being 0.625 and specificity being 0.569 ^[17]. In the meta-analysis of diagnostic accuracy of PSA for the detection of PCa in 14,489 symptomatic patients and a control group of 9459 men (normal DRE and PSA <4 ng/mL for 7 years), pooled data found the sensitivity of PSA to be 0.93, with a specificity of 0.20, for symptomatic patients, confirming its known high sensitivity but very poor specificity [18,19]^[18][19].

PHI performs as a promising diagnostic biomarker with enhanced specificity and accuracy in detecting PCa, especially its clinically significant forms. By incorporating PHI into clinical practice, current diagnostic gaps could be diminished, offering patients more accurate screening and subsequently better treatment choices.

2.3. 4Kscore

The 4Kscore (developed by OPKP Health, Inc.) is a follow-up blood test performed if the patient has abnormal PSA and DRE results, to detect indolent disease and prevent unnecessary biopsies. This test analyzes four prostate-specific biomarkers (total PSA, free PSA, intact PSA, and human kallikrein 2 [hK2]), along with the patient’s clinical information (age, biopsy history, DRE result) via a complex algorithm to produce a final score. Several studies confirmed the usefulness of the abovementioned biomarkers for PCa prediction. In a study involving 161 patients scheduled for radical prostatectomy, hK2 levels correlated positively with the pathologic stage of clinically localized PCa ^[20]. Similarly, serum specimens from 577 patients, treated with radical prostatectomy, showed that increased concentrations of hK2 in the blood were significantly associated with unfavorable features of PCa ^[21]. A different study emphasized hK2’s superiority over PSA in accurately identifying poorly differentiated (G3) tumors, which is paramount for clinical decision-making ^[22]. Sera from 122 patients with PCa, graded from G1 to G3, was studied for total PSA, free PSA, and hK2 readings. hK2 significantly improved the identification of poorly differentiated (G3) tumors compared with PSA, ultimately allowing for better clinical decision-making ^[22].

The 4Kscore test is approved by the FDA and included in current USA, Canadian, and European PCa early-detection guidelines. The accuracy of the test was demonstrated in several validation studies. In a study of 1012 men scheduled for prostate biopsy, the 4Kscore showed better diagnostic performance in detecting significant PCa than standard of care, with an AUC of 0.82, and the authors stated that up to 36% of biopsies could be avoided or delayed ^[23]. In a prospective trial of 366 enrolled men (56% African American, 36% Grade Group 2 or higher), the 4Kscore test showed better discrimination (AUC 0.81 versus 0.74) and clinical usefulness than the base model ^[24]. In a nested case-control study of 12,542 men, followed for >15 years, PSA and 4Kscore were measured to increase the specificity of screening for lethal PCa at an early stage. The 4Kscore significantly enhanced the prediction of metastasis compared with PSA alone (PSA > 2 ng/mL). ^[25]. A study involving 11,506 men followed for 20 years was undertaken to determine the long-term risk of PCa mortality using the 4Kscore. In men with elevated PSA (≥2.0 ng/mL), predictive accuracy was improved by the 4Kscore compared with PSA alone (0.80 versus 0.73). Also, it was determined that men with an elevated PSA but a low 4Kscore can be put under surveillance rather than undergoing invasive biopsy ^[26].

The 4Kscore test proves to be a useful tool in deciding if a patient needs to go through a biopsy procedure after repeated abnormal PSA readings and abnormal DRE findings (Table 1).

3. Urine-Based Biomarkers

Urine is a promising liquid biopsy choice, especially for PCa, because prostate manipulation enriches it with PCa cells, exosomes, DNAs, RNAs, proteins, and small molecules, which could be used as biomarkers. In comparison with biopsy cores, urine sampling provides a more representative picture of the significant spatial heterogeneity within the patient’s prostate. Several urine-based tests are available: the PROGENSA PCA3 assay, the MiProstate Score assay, the ExoDx™ Prostate IntelliScore test, and the SelectMDx test (Table 1). Even though post-DRE or DRE urine specimens are boosted with prostate secretions, and therefore preferable for following prostate biomarker analysis, non-invasive, non-DRE urine-based gene panels are emerging, with the ExoDx™ Prostate Test being CLIA-approved [27,28,29,30]^{[27][28][29][30]} (Table 2).

4. Additional Biofluid Markers in Development

Table 3 summarizes examples of promising methylation and RNA-based urinary biomarkers in development.

Table 3.

Examples of promising methylation and RNA-based urinary biomarkers in development.

Molecular Target	Patient Cohort, N	Sensitivity %	Specificity%	AUC	Reference
24 gene methylation panel	104	100	97	0.88–0.99	[56]^[31]
miR-34b/c, mir193b methylation	161	97	80	0.98	[57]^[32]
RARβ2 methylation, meta study	1181	-	-	-	[58]^[33]
APC, CRIP3, GSTP1, HOXD8 methylation	153	-	-	-	[59]^[34]
HOXD3, GSTP1 methylation	408	57	97	0.8	[60]^[35]
miR-148a, miR-375	277	83	79	0.79–0.84	[61]^[36]
miR-21, miR-375, 5 miRNA panel	70–215	-	-	0.87	[62,63]^[37][38]
miR-222-3p/miR-24-3p/miR-30c-5p	249–1047	-	-	0.71–0.95	[64,65]^[39][40]
PSGR	146–215	-	-	0.68–0.90	[66,67]^[41][42]
PCGEM1, PCA3	271	-	-	0.88	[68]^[43]
PSMA, PSGR, PCA3	154	-	-	0.82	[69]^[44]

PSGR, a gene with homology to the G protein-coupled odorant receptor family, has been identified as a PCa-specific gene, which is overexpressed in PCa but shows very little or no expression in prostatic epithelial cells of matched normal specimens. Overexpression of PSGR in PCa provides a foundation for the potential of PSGR as a PCa biomarker [70,71]^[45][46]. It was found that, compared to normal and BPH tissues, the expression of PSGR increased significantly in PIN and prostate tumors (approximately 10-fold), especially in early prostate cancer, suggesting PSGR has the potential for early PCa detection (AUC of 0.902) [66]^[41]. PSGR expression was readily detected by qPCR in post-DRE urine sediments of 215 patients for PCa detection [67]^[42]. A total of 220 RNA specimens from benign and malignant prostatic epithelial cells of 110 PCa patients were investigated for association of the PSGR overexpression in the different PCa pathologic stages. PSGR overexpression showed a positive correlation with pT3 and a higher level of serum PSA. Additionally, PCa cells of African American patients demonstrated about a two-fold increase of PSGR expression in comparison to Caucasian American patients [72]^[47]. A prostate-specific gene, PCGEM1, was identified as long non-coding RNA, preferentially expressed in prostate tumors [73]^[48], and PCGEM1 expression was significantly higher in the PCa of African American men than in Caucasian American men [74]^[49]. PCGEM1 expression was found to be markedly downregulated following castration and upregulated upon androgen receptor activation in vivo. PCGEM1 expression levels were repressed in response to androgen ablation therapy, indicating that PCGEM1 is a transcript regulated by androgens [75]^[50]. In another study focusing on non-DRE urine specimens, collected from patients undergoing diagnostic biopsy, a two-gene panel, consisting of PCGEM1 and PCA3, was used to differentiate high-grade cancer (Gleason score 7–10) from low-grade cancer (Gleason score 6) in a patient cohort with about 30% of African American men. When combined with standard of care variables, this panel notably enhanced the prediction of high-grade cancer at diagnosis compared to using standard of care variables alone (AUC 0.88 vs. 0.80) [68]^[43]. Prostate-specific membrane antigen (PSMA) is a transmembrane protein predominantly found in PCa epithelial cells, exhibiting higher expression levels in cancerous cells than in non-cancerous cells. In a study investigating post-DRE urine samples from 154 patients undergoing prostate biopsies, biomarkers consisting of PSMA, PSGR, and PCA3 were used to distinguish between advanced versus indolent PCa, to then determine which patients should undergo biopsy. It was concluded that the PSMA, PSGR, and PCA3 scores were significant indicators of PCa, with an AUC of 0.82 [69]^[44]. In a further investigation, the diagnostic relevance of five PCa-associated genes (PSMA, Hepsin, PCA3, GalNAC-T3, and PSA) was evaluated in 44 patients diagnosed with PCa, and 46 patients with BPH were reviewed along with post-DRE urine samples. After measuring the absolute concentration of each gene’s transcript, the diagnostic capabilities of the combined urinary PSA and PSMA levels were more effective than any singularly considered marker [76]^[51].

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