Current and emerging tissue-based molecular biomarkers for prostate cancer management: A narrative review

Abstract

Prostate cancer is a heterogeneous disease process with a wide spectrum of clinicopathologic variables that impact diagnosis, risk stratification, and management. To improve diagnostic accuracy and to better inform clinical decision making, the development of molecular biomarkers has undergone considerable discovery and clinical validation in the past decade. Prostate cancer is no longer seen as a single disease entity but one with considerable heterogeneity existing between tumors and between patients. Biomarkers now allow for more personalized and precision-based approaches to management that otherwise would have depended on applying clinical algorithms alone. The aim of this review is to discuss and evaluate prostate cancer tissue-based biomarkers that have been developed to aid diagnosis, improve risk stratification, and management.

Keywords

biomarkers / biopsy / precision medicine / prostate cancer

1 INTRODUCTION

Prostate cancer risk stratification and clinical decision making is hampered by substantial rates of pathological misclassification and upgrading following radical prostatectomy (RP). While risk stratification based on clinicopathologic variables alone can provide a reasonable means for patient counseling and expectations regarding clinical outcomes, prognostication using prostate-specific antigen (PSA) levels and Gleason score alone is often insufficient and improved prognostic markers are gravely needed[1]. For this reason, there have been several tissue-based biomarkers that have undergone development and clinical validation in the past decade.

Various tissue-based molecular biomarkers can be utilized in the diagnostic, prognostic, and predictive management of prostate cancer. According to the American Society of Clinical Oncology, the goal of a biomarker is to “estimate the likelihood of a disease characteristic being present or absent, more accurately determine prognosis, or provide the probability of response to a specific treatment”[2]. These biomarkers can help inform clinical decision making regarding the likelihood of a patient harboring a more indolent pathologic subtype that would favor active surveillance (AS) versus more aggressive subtypes that would favor primary treatment with RP or radiation therapy. In addition, biomarkers may help predict the likelihood of metastatic spread following primary therapy, as seen in patients with germline mutations (e.g., BRCA1, BRCA2, MSH). Currently, no clinical guidelines recommend these tests in routine clinical practice except when they are combined with other clinicopathologic variables, which may help the clinician and patient decide if a certain treatment pathway is better indicated.

Biomarkers specific to prostate cancer can broadly be categorized according to the stage in the patient's overall course of diagnosis and treatment. For example, some biomarkers are intended for assessing whether a patient should undergo a prostate biopsy while others are intended to inform decision making between AS or treatment. Other tests are intended to inform patients of the likelihood of having advanced disease or disease at risk of eventual progression to metastasis, which may help patients and clinicians decide regarding post-RP adjuvant or early salvage therapy. Finally, it is expected that biomarkers will help reduce overdiagnosis, more accurately stratify patients based on cancer subtype, improve treatment allocation, and improve patient quality of life and life expectancy[3].

2 PROSTATE CANCER BIOMARKER DISCOVERY

The mechanisms underlying carcinogenesis are multifactorial, however, genetic alterations in transcriptional and epigenetic profiles underpin the development of cancer in otherwise normal cells. Numerous genetic biomarkers have been identified across the entire human genome that have been implicated in the development of various cancers including oncogenes and tumor suppressor genes. These biomarkers serve as useful targets both for the development of diagnostic tools and as potential actionable therapeutic targets. Given the vastness of the human genome, identifying novel biomarkers in this manner can be time consuming and labor intensive. To expedite this process, high-throughput massive sequencing techniques have been developed[4].

Next-generation sequencing (NGS) is a high-throughput sequencing technology that allows for the rapid sequencing of thousands of genetic sequences in parallel with a single assay. Novel molecular cancer biomarkers may be discovered this way, as it allows for the rapid detection of point mutations, insertions, deletions, copy number variations, translocations, fusion genes, differential gene expression, and differential messenger RNA (mRNA) splicing. This technology is particularly useful for analyzing tissue from different patients in whom tumor heterogeneity exists where the genomic composition of each tumor may be different even within the same patient. This allows for a personalized approach to prostate cancer risk stratification and treatment decision making[5]. In 2019, Ikeda et al. reported on the use of NGS in the identification of unique genomic alterations in prostate cancer. They found that prostate cancer harbored 148 unique alterations affecting 63 distinct genes. The median number of genomic alterations per patient was three, with a range of one to nine. The most common genomic alterations were identified in TP53 (55.2%), PTEN (29.9%), MYC (17.9%), PIK3CA (13.4%), APC (9.0%), BRCA2 (9.0%), CCND1 (9.0%), and RB1 (9.0%). A small number of patients contained abnormalities in BRAF, NTRK, and ERBB2[6]. While the rate of use of clinical-grade NGS remains low and has been employed in only a minority of clinical trials, this technology offers immense promise as a tool for identifying novel biomarkers.

3 SERUM-BASED BIOMARKERS

3.1 Prostate specific antigen (PSA)

PSA is a serine protease coded by the kallikrein 3 (KLK3) gene on chromosome 19q 13.1–13.4. It is produced by ductal and acinar epithelium in the prostate. PSA exists in protein-bound (e.g., α-1-antichymotrypsin) and free molecular forms referred to as free PSA (fPSA)[7]. Protein bound and fPSA together comprise total PSA (tPSA)[3]. Approximately 65%–95% of tPSA is protein bound. The clinical usefulness of this distinction lies in calculation of the free-to-total PSA ratio, where low ratio values have been identified as being more predictive of a prostate cancer diagnosis[8]. PSA was initially approved by the United States Food and Drug Administration (FDA) in 1986 to monitor disease progression in men already diagnosed with prostate cancer and only later in 1994 received approval for the diagnosis of prostate cancer, along with the digital rectal examination (DRE)[9]. It is important to note that PSA elevation may be attributed to nonmalignant etiologies as well including benign prostatic hyperplasia, prostatitis, sexual intercourse, and recent urinary tract instrumentation[10].

3.2 Prostate Health Index (PHI)

The PHI (Beckman Coulter, USA) is a mathematical composite test that analyzes fPSA, tPSA, and precursor PSA ([-2]proPSA, p2PSA) to improve the accuracy of detecting clinically significant prostate cancer, specifically Gleason ≥ 7 prostate cancers at biopsy[11]. TPSA and fPSA alone have limited specificity to detect clinically significant, curable prostate cancer and therefore the addition of [-2]proPSA has been evaluated to see if this combination improves predictive accuracy. The PHI has been demonstrated to outperform its individual components (particularly for the prediction of high-grade clinically significant prostate cancer) and is approved for use in men over age 50 with a nonsuspicious DRE and serum PSA of 4–10 ng/mL[12]. It was first approved by the US FDA in 2012.

In a prospective, multi-institutional trial of 892 men with no history of prostate cancer, normal DRE, and PSA between 2 ng/mL and 10 ng/mL, a six-core or greater prostate biopsy was performed. In the 2–10 ng/mL range, the PHI had an 80% and 95% sensitivity and specificity, respectively. Increasing PHI was associated with a 4.7-fold increased risk of prostate cancer and a 1.61-fold increased risk of Gleason score ≥ 7 on biopsy. The area under the curve (AUC) of the PHI performed better than free-to-total PSA (0.724 vs. 0.670) with respect to distinguishing clinically significant prostate cancer from low-grade disease or a negative biopsy[12]. Similar results were reported by Loeb et al., wherein PHI was confirmed to outperform its individual components in terms of the prediction of overall and high-grade cancer on biopsy. It has also been shown to predict the likelihood of progression during AS[13].

3.3 4KScore

The 4KScore® Test (OPKO Health, USA) is another composite test that combines the four kallikrein proteins including tPSA, fPSA, intact PSA, and human kallikrein 2. By combining the 4KScore with clinical data such as age, DRE, and history of prior negative biopsy, the likelihood of both a positive cancer diagnosis on subsequent biopsy and the likelihood of aggressive disease can be obtained[14]. The test is indicated in men aged 45 years and older with an abnormal age-specific tPSA and/or abnormal DRE, to assess the need for prostate biopsy. A 4KScore less than 5.0 is associated with a low risk of Gleason ≥ 7 prostate cancers. The test received US FDA approval in 2021[2].

Using blood samples obtained from 943 men included in the Swedish Cancer Registry, Vickers et al., assessed the performance of the 4KScore to predict subsequent prostate cancer diagnosis in men with a total PSA of 3.0 ng/mL or higher at baseline. The full kallikrein panel was found to enhance the predictive accuracy for clinically diagnosed prostate cancer. They concluded that men with a PSA of 3.0 ng/mL or higher, who are otherwise defined as low risk based on the four kallikrein panel, are highly unlikely to develop incurable prostate cancer[15].

The test can also be used to predict the likelihood of developing distant metastases in men with a PSA of ≥ 2 ng/mL. Stattin et al., measured the four kallikreins in cryopreserved blood from a population-based cohort in Sweden including 12 542 subjects who had been followed for more than 15 years. For men with PSA > 2 ng/mL, the four kallikrein model significantly enhanced the prediction of metastasis compared with PSA alone. Approximately 50% of men with PSA > 2 ng/mL were defined as low-risk based on this model and had a 15-year risk of metastasis of ≤1%[16].

The predictive accuracy and economic impact of the four kallikrein panels were investigated in a meta-analysis including 8500 patients, of which 2780 were found to have prostate cancer and 598 of those individuals had high-grade prostate cancer. The analysis demonstrated a statistically significant improvement in predictive accuracy of 8%–10%. Also, it was noted that the panel could reduce unnecessary biopsies by 48%–56%, potentially translating into an annual savings of close to $1 billion in the United States[17].

3.4 Androgen receptor splice variant 7 (AR-V7)

AR-V7 is a splice variant of the AR mRNA with resultant truncation of the ligand-binding domain of the full-length AR. While AR-V7 mRNA levels have been detected in normal prostate tissue, much higher expression levels have been reported in prostate tumors with an even higher level in tumors from patients who eventually developed castration resistance compared with those in hormone-naïve tumor specimens[18]. In addition, AR-V7-associated antibody testing has demonstrated that in hormone-naïve prostate cancer cells, AR-V7 is specifically confined to the cytoplasm, whereas it exhibits nuclear translocation following castration resistance development[19].

In 2019, Sharp et al., reported on a novel AR-V7 antibody that was used to evaluate AR-V7 protein expression, immunohistochemically, in 358 primary prostate samples and 293 metastatic biopsies. They found that the AR-V7 protein is rarely expressed (<1%) in primary prostate cancer, but is highly expressed (75%) in tumors subjected to androgen deprivation therapy and even higher levels after exposure to abiraterone acetate or enzalutamide therapy. Consistent with the results reported by Guo et al., AR-V7 expression was nuclear in 94% of cases of metastatic castration-resistant prostate cancer (mCRPC). Finally, they demonstrated that AR-V7-negative disease is associated with better PSA response and overall survival (74 months for AR-V7-negative vs. 25 months for AR-V7-positive) in patients being treated with endocrine therapies[20].

The expression of AR-V7 in circulating tumor cells (CTCs) is associated with poor outcomes in patients being treated with AR axis-targeted therapies (AATTs) due to increased rates of resistance to therapy[21]. Specifically, these patients have reduced progression-free survival and overall survival[22]. This biomarker is particularly useful in patients with mCRPC as AR-V7 positivity is associated with a poor response to AATT and therefore may help direct patients toward chemotherapy or other non-AATT systemic therapy[23].

The detection of the AR-V7 splice variant is conducted through the use of liquid biopsy, which is essentially a test of the peripheral blood to detect CTCs. The liquid biopsy is advantageous for being minimally invasive, easier than focal metastatic lesion biopsy, and can provide an “average” of AR-V7 expression in a patient due to the heterogeneity of AR-V7 expression at different metastatic sites[21]. The CellSearch CTC (Menarini Group, Italy) is a clinically validated, minimally invasive liquid biopsy assay that is FDA approved for predicting overall survival and progression-free survival in metastatic prostate cancer[24]. Specifically, >5 CTCs are associated with poor overall outcomes. Moreover, changes in CTC levels can be used to assess early treatment response[25].

Commercially available assays to asses for CTCs may be designed to either detect intranuclear AR-V7 protein (Oncotype DX AR-V7 Nucleus Detect Test by Genomic Health/Epic Sciences, USA) or AR-V7 mRNA using a polymerase chain reaction (PCR)-based assay (Qiagen AdnaTest, USA). In a sample of 181 patients with mCRPC, 227 peripheral blood samples were tested for AR-V7 status on CTCs using the AdnaTest. These were then matched with mCRPC biopsies from the same patients. AR-V7 positivity was noted on 35% of the samples while another 31% had detectable CTCs but were AR-V7 negative with the remaining 35% of samples having no detectable CTCs. The AR-V7 CTC-positive group exhibited more advanced disease, higher disease burden, worse performance status, higher number of prior taxanes therapies received, lower hemoglobin levels, higher alkaline phosphatase levels, higher lactate dehydrogenase levels, and higher PSA levels compared with the CTC-positive/AR-V7-negative and CTC-negative groups[20].

The PROPHECY trial was a multicenter, prospective-blinded study of 118 men with high-risk mCRPC starting either abiraterone acetate or enzalutamide therapy. The study utilized the AdnaTest CTC AR-V7 mRNA assay and the Epic Sciences CTC nuclear-specific AR-V7 protein assay in a head-to-head comparison. After adjusting for CTC number and clinical prognostic factors, AR-V7 detection was associated with a shorter radiographic progression-free survival and overall survival. The observed percentage agreement between the assays was 82%[26]. The results confirmed AR-V7 positivity, which was demonstrated by either assay and was predictive of a lack of benefit to AATT.

The impact of AR-V7 results on treatment decision making was investigated by Markowski et al., who conducted a questionnaire-based survey of 38 providers in Canada and the United States. The providers ordered tests on a total of 150 patients. For 53% of the patients, the treatment decision had been modified based on the results of the AR-V7, specifically for AR-V7-positive patients in whom the treatment was altered in 86% of the patients[27]. These results support the clinical use of these assays in the evaluation and management of patients with mCRPC and are highly likely to influence treatment decision making.

Table 1 summarizes the various serum-based biomarkers according to molecular marker, validation and prognostic data, and FDA approval status.

4 URINE-BASED BIOMARKERS

4.1 Prostate cancer antigen 3 (PCA3)

The PCA3 is a nonprotein coding RNA that has been found to be overexpressed in essentially all prostate cancers. The antigen was first identified by Bussemakers et al., in 1999 as the DD3 gene, now known as PCA3[28]. PCA3 levels are evaluated in post-DRE urine specimens with the value being independent of prostate volume or the presence of other prostatic diseases (e.g., prostatitis). PCA3 RNA levels have been shown to be significantly more predictive of clinical outcomes following prostate biopsy compared with serum PSA[29].

The Progensa PCA3 assay (Hologic, USA) is a PCR-based molecular assay that detects the overexpression of PCA3 mRNA in urine and is indicated in men with an elevated serum PSA and prior negative biopsy results as a decision tool to assess if a repeat biopsy is indicated[30,31]. The test was first approved for use by the US FDA in 2012. The test examines the ratio of PCA3 RNA, a prostate cancer-associated RNA with PSA RNA in patient urine, followed by DRE[32]. In a meta-analysis of 12 295 patients with prostate cancer, the pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, and AUC were 0.65, 0.73, 2.23, 0.48, 5.31, and 0.75, respectively[33].

4.2 ExoDx Prostate IntelliScore (EPI)

The EPI is a urine exosome genome expression-based assay that measures ERG and PCA3, with SPDEF as an internal reference. The assay utilizes non-DRE urine to assess for the risk of Gleason 6, Gleason 7, and benign prostate disease on biopsy in men aged 50 years and older and with PSA from 2 ng/mL to 10 ng/mL. Phase I and II validation testing has confirmed the ability of the test to distinguish Gleason grade 2 (GG2) and higher cancer from GG1 cancer in men 50 and older with PSA from 2 ng/mL to 10 ng/mL[3,34]. Previous studies have reported negative predictive values of 91% for ≥GG2 and 97% for ≥GG3 disease. The test can be used to discriminate between the risk of no cancer/low-grade prostate cancer and high-grade prostate cancer with an EPI score of ≤15.6 suggestive of GG1 disease and a score >15.6 suggestive of ≥GG2 disease[34].

Tutrone et al. evaluated the impact of the EPI test on decision to biopsy in a real-world clinical setting using a prospective, randomized, blinded, two-armed clinical utility study of 1094 patients. All patients received the EPI test but only those in the EPI arm received their results compared with controls. Overall, the study demonstrated that the test did influence decision making with respect to deferring or proceeding with biopsy and that the results of the EPI test led to the detection of 30% more high-grade prostate cancers compared to the control arm. Of the urologists in the study, 68% reported that the test result ultimately influenced their decision to defer or proceed with biopsy[35].

4.3 SelectMDx

SelectMDx (MDxHealth, USA) is a quantitative real-time PCR assay performed on urine specimens obtained after DRE. The test is used to determine the probability of a patient having high- or low-grade prostate cancer on prostate biopsy. The assay measures the mRNA levels of the DLX1 and HOXC6 genes. This test has been shown to improve the detection of clinically significant prostate cancer compared with PSA and PCA3[36].

Van Neste et al. developed a multimodal model that incorporates mRNA biomarkers and traditional risk factors to identify patients with high-grade prostate cancer. They conducted two prospective multicenter studies in which post-DRE urine was collected and mRNA profiling was performed to develop a risk score that was first developed using a 519-person cohort and then clinically validated in an independent cohort of 386 men. They found that HOXC6 and DLX1 were good predictors for the detection of high-grade prostate cancer with an AUC of 0.90 in the validation cohort. The model was shown to be superior to the Prostate Cancer Prevention Trial risk calculator and PCA3 assay in improving clinical decision making and reducing the performance of unnecessary biopsies[37].

5 PTEN/TMPRSS2:ERG

The PTEN/TMPRSS2:ERG is an assay developed and distributed by Metamark (USA). This assay detects the presence of PTEN and the fusion gene product of TMPRSS2 and ERG. PTEN is a tumor suppressor gene and its deletion is associated with an increased risk of high-grade prostate cancer and tumor progression. PTEN codes for a lipid phosphatase that opposes the oncogenic PI3K/AKT pathway. PTEN loss results in disruption of the PI3K signaling pathway, which is responsible for controlling cellular proliferation and growth[38]. Increasing levels of PTEN loss have been linked to higher GG groups[39]. The degree of PTEN loss has also been shown to correlate with clinical stage where PTEN loss of approximately 20% has been reported in the primary tumor, while rates of 40% loss have been reported in metastatic tumors[7]. The clinical impact of PTEN loss was investigated on 107 prostate cancer specimens using fluorescence in situ hybridization. PTEN loss was associated with an earlier onset of B-cell receptor (BCR), while homozygous PTEN loss was associated with an even earlier time to BCR. In addition, PTEN loss at the time of RP was associated with a higher likelihood of extraprostatic extension and seminal vesicle invasion[40]. Using a PTEN immunohistochemical assay performed on tissue microarrays, and after adjusting for clinicopathologic variables, PTEN loss (complete or heterogeneous) was found to be associated with lethal progression. Interestingly, this risk was only found among men who were in the ERG-negative group and not in the ERG-positive group[41].

The fusion of the AR-regulated TMPRSS2 gene promotor and the N-terminally deleted ETS transcription factor, ERG, represents the most common prostate cancer-specific driver gene alteration[3]. These gene fusions have been found in 50%–65% of patients of European ancestry with prostate cancer. The potential of urinary ERG to improve prostate cancer detection was tested in a cohort of 237 patients who provided post-DRE urinary samples prior to scheduled prostate biopsy. Urinary ERG mRNA was measured and an ERG score was reported. In total, 40.9% of the subjects had prostate cancer on biopsy and a higher urinary ERG score was associated significantly with malignancy on biopsy but not with clinical stage or Gleason score. The urinary ERG score performed the best in Caucasian men and in those with a PSA of ≤4 ng/mL[42].

Researchers from the University of Michigan have developed a post-DRE-based urinary test called the MyProstateScore. The test is a validated, locked multivariable model that combines urinary PCA3, urinary TMPRSS2:ERG scores, and serum PSA. In a cohort of 1525 biopsy-naïve men, 338 were found to have grade group ≥2 cancer on biopsy. Using a MyProstateScore threshold of 10, the sensitivity and negative predictive value for grade group ≥2 cancer were 97% and 98%, respectively. At a threshold score of ≤10, the test would have prevented 32% of unnecessary biopsies while only missing 3.5% of grade group ≥2 cancers[43].

To develop a clinical algorithm to help direct the need for prostate biopsy in patients with PSA < 10 ng/mL, Salami et al. examined the post-DRE urine sediment from 45 patients, prior to prostate biopsy at two centers. The performance of TMPRSS2:ERG fusion, PCA3, and serum PSA for the prediction of prostate cancer on biopsy was analyzed. TMPRSS2:ERG on post-DRE urine was associated with prostate cancer (odds ratio [OR] = 12.02, p < 0.05), while PCA3 had the highest sensitivity in predicting prostate cancer at 93%. TMPRSS2:ERG had the highest specificity and greatest discriminatory value for predicting prostate cancer, compared with PCA3 and serum PSA alone. When serum PSA, PCA3, and TMPRSS2:ERG were all combined, the clinical utility for cancer prediction improved with a sensitivity and specificity of 80% and 90%, respectively[44].

Table 2 summarizes the various urine-based biomarkers according to molecular markers, validation and prognostic data, and FDA approval status.

6 PROSTATE TISSUE-BASED BIOMARKERS

6.1 Decipher

The Decipher microarray test (Decipher Biosciences, USA) utilizes RNA obtained from formalin-fixed, paraffin-embedded prostate tissue following prostate or RP. The Decipher score is reported between 0 and 1 with 0–0.45 indicating low-risk disease, 0.46–0.60 indicating average-risk disease, and 0.61 and higher indicating high-risk disease. The test measures the expression level of 22 different genes including LASP1, IQGAP3, NFIB, S1PR4, THBS2, ANO7, PCDH7, MYBPC1, EPPK1, TSBP, PBX1, NUSAP1, ZWILCH, UBE2C, CAMK2N1, RABGAP1, PCAT-32, GLYATL1P4, PCAT-80, and TNFRSF19[46]. These genes are involved in processes such as cellular proliferation, differentiation, cell cycle progression, and AR signaling. The assay provides an assessment of the risk of high-grade pathology following prostatectomy, the 5-year risk of metastasis, and the 10-year risk of prostate cancer-specific mortality. The goal of the test is to help inform the use of salvage versus early adjuvant radiotherapy following prostatectomy, however, the test has not been prospectively validated in clinical studies[47].

The Decipher test was first developed using postradical prostatectomy specimens from the Mayo Clinic where tumors from 639 patients were reviewed. A genomic classifier was developed using differential RNA expression of 1.4 million separate genomic markers in men found to have rising PSA after prostatectomy including those with early metastasis after BCR. The 22 gene classifier was developed and then validated in two separate cohorts where higher genomic classifier scores were associated with earlier death after prostate cancer and reduced overall survival[46]. Later studies eventually validated the test for use in predicting metastasis and prostate cancer-specific survival[48,49].

6.2 Prolaris

Prolaris is a molecular score assay that is based on the expression of 31 cell cycle progression (CCP) genes that are involved in cancer proliferation. There are two separate tests that can be performed based on whether the specimen is obtained from prostate biopsy (Prolaris biopsy test) or following RP (Prolaris postprostatectomy test). The Prolaris biopsy test provides information regarding the 10-year prostate cancer-specific mortality and the risk of 10-year metastasis with definitive treatment. The Prolaris postprostatectomy test provides information on the risk of BCR at 10 years postsurgery. The test is scored from 0 to 10 with a higher score indicative of more aggressive cancer and a higher rate of disease progression[50]. Cuzick et al. first clinically validated this 31 gene signature in two cohorts of patients, including 366 patients who had undergone prostate biopsy and 337 men who were diagnosed with localized prostate cancer following transurethral resection. The authors found that the CCP score was associated with BCR after definitive surgery and the risk of prostate cancer-specific mortality in the conservative treatment group[51]. In a separate study of 413 men, 82 had experienced either BCR or had undergone salvage treatment. After controlling for clinical and pathologic variables, the CCP score was assessed with respect to predicting recurrence. The CCP score was validated to have significant prognostic accuracy and may provide a valuable tool to identify men with BCR who may benefit from earlier adjuvant therapy[52].

Based on results from the PROCEDURE-1000 study, which was a large, prospective registry with about 1600 patients, the CCP score contributed to a change in treatment in 47.8% of patients including de-escalation of treatment in 75% of cases and escalation in the other 25%[53]. The Prolaris test is recommended by the National Comprehensive Cancer Network (NCCN) in men with very-low-, low-, and favorable intermediate-risk prostate cancer on biopsy with a life expectancy of at least 10 years[50].

6.3 Oncotype DX

The Oncotype DX assay is developed and distributed by Genomic Health (USA). The genomic assay utilizes reverse-transcriptase PCR to measure the expression levels of 17 genes involved in various cellular pathways regulating proliferation, AR signaling, cellular organization, and stromal response. The 12 cancer-specific genes include TPX2, AZGP1, KLK2, SRD5A2, FAM13C, FLNC, GSN, TPM2, GSTM2, BGN, COL1A1, and SFRP4[50]. The end output of the assay is a Genomic Prostate Score (GPS), which produces a score based on the expression levels of the genes, that range from 0 to 100. Higher scores are associated with a higher likelihood of adverse pathology such as a higher GG group or having locally advanced, nonorganized disease following RP[54]. The GPS is the only commercially available test to provide key prostate cancer end points including the risk of high-grade (≥GG group 3) disease, risk of pT3+ disease, risk of metastasis within 10 years, and the risk of prostate cancer-specific mortality within 10 years[55]. The assay also provides information regarding the likelihood of adverse pathology at the time of prostatectomy and the likelihood of disease progression[56].

The Oncotype DX GPS assay was initially validated for use in patients with breast and colon cancer and later underwent validation and approval for use in prostate cancer. Klein et al. performed a large validation study in which 732 candidate genes were first established for exploration. Of the 732 genes, 288 (39%) were identified to be predictive of clinical recurrence, despite tumor heterogeneity and multifocality. Another 198 genes were found to be predictive of aggressive disease after adjusting for clinicopathologic variables including PSA, Gleason score, and clinical stage. Further analysis led to the development of the 17 multigene expression-based signature. The authors then performed an independent clinical validation study on 395 retrospectively collected needle biopsies from men with low to intermediate clinical risk prostate cancer who were candidates for AS. Following validation, the GPS was demonstrated to be predictive of high-grade and high-stage pathology following RP[55].

The ability of the Oncotype DX GPS to predict clinical outcomes following primary treatment was reported by Cullen et al.[56] Prostate biopsy specimens from 431 men with NCCN classified very-low-, low-, or intermediate-risk prostate cancer were tested with the multigene signature to determine if GPS score was associated with the risk of adverse pathology, BCR, and metastatic recurrence. GPS results were obtained in 402 cases, of which 62 men had BCR, five developed metastases, and 163 demonstrated adverse pathology. The GPS was found on univariable analysis to be predictive for BCR, time to metastatic recurrence, and was strongly associated with the risk of adverse pathology. The median GPS score was 30.3 for both African American and Caucasian men[56]. The results of this study highlighted the ability of the GPS to predict cancer aggressiveness and help both patients and clinicians decide if proceeding to immediate treatment is beneficial, particularly for those considering or undergoing AS.

The ability of the Oncotype DX GPS test to predict adverse pathology (Gleason score ≥4 + 3 and/or ≥pT3) at the time of RP was investigated by Eggener et al., using 1200 men in a multi-institutional prospective study. In total, 114 patients underwent prostatectomy and 40 (35%) demonstrated adverse pathology. The GPS was a significant predictor of adverse pathology in univariable analysis and continued to be significant after controlling for clinicopathologic variables such as Gleason score, clinical T-stage, PSA, and NCCN risk group. The authors also examined the effect of the GPS on patient and physician attitudes regarding clinical decision making using the Decisional Conflict Scale. Compared with the pre-GPS scale score (27), there was a significant improvement after GPS with a post-GPS scale score of 14. This study further confirmed the ability of the Oncotype DX GPS to serve as a biomarker for adverse pathology prediction while improving patient decision-making ability[57].

The impact of the Oncotype DX GPS test to influence changes in treatment recommendations was investigated by Badani et al. This was a prospective study performed at three high-volume urology practices in men with specific NCCN criteria. The treatment recommendation was assessed before and after the GPS. In total, 158 men were included in the analysis, which showed that the treatment changed between AS and immediate treatment in 18% of the subjects, after the GPS. With respect to AS, there was a relative increase in recommendation in 24% of subjects. The treatment modality and/or intensity changed after GPS in 41/158 men (26%). Of the 41 men, 25 underwent a decrease in recommendation intensity, 14 underwent an increase, and remained unchanged in two. In patients who were initially recommended for RP, 30% underwent a change in recommendation to AS. The results of this study have provided support for the ability of the test to enhance surgeon confidence in their clinical treatment recommendations[58].

6.4 ConfirmMDx

ConfirmMDx is developed and distributed by MDxHealth (USA). This is a noninvasive tissue-based prognostic epigenetic assay performed on previously obtained negative prostate biopsy specimens. The test is used to assess the risk of harboring prostate cancer nearby in men with a prior negative prostate biopsy who may have a persistently abnormal PSA and who would like to know if a repeat biopsy should be considered[23]. This multiplex assay provides quantification of the DNA methylation of promotor genes on three distinct prostate cancer-associated tumor suppressor genes including glutathione S-transferase Pi 1 (GSTP1), Ras association (RalGDS/AF-6) domain family member 2 (RASSF2), and Adenomatous Polyposis Coli (APC) using PCR. CpG island expansion has been correlated with an increased risk of prostate cancer development in benign prostate biopsy samples due to epigenetic alterations[59,60]. An important point of consideration in the use of ConfirmMDx and the studies that evaluated its utility is that the trials were conducted prior to the routine use of prostate magnetic resonance imaging (MRI)[50].

The first study that evaluated the utility of this three-gene assay panel was the Methylation Analysis to Locate Occult Cancer (MATLOC) study, which evaluated prostate biopsy needle core tissue samples from 498 men from the United Kingdom and Belgium who had histopathologically negative prostate biopsy specimens and subsequently underwent repeat biopsy within 30 months. The results demonstrated that the assay has a sensitivity and specificity of 68% and 64%, respectively. Also, the negative predictive value was 90% (95% confidence interval [CI]: 87–93) and when controlling for clinicopathologic variables such as patient age, PSA, DRE, and first biopsy histopathology, the assay was a significant independent predictor of patient outcome[61].

The second study was the multicenter Detection of Cancer Using Methylated Events in Negative Tissue (DOCUMENT) study, which evaluated prostate cancer-negative prostate biopsy core tissue samples from 350 men from five urological centers in the United States. All of the patients underwent a repeat prostate biopsy within 24 months and were blindly evaluated on pathologic review of both series of biopsies. The epigenetic assay was then performed using quantitative methylation-specific PCR. The results demonstrated a negative predictive value of 88% (95% CI: 85–91). Similar to the MATLOC study, multivariable analysis after correcting for clinicopathologic variables, similar to the ones mentioned previously, demonstrated that the assay was the most significant independent predictor of patient outcome[62].

Van Neste et al. took the evaluation further with their study assessing the predictive ability of the epigenetic assay in detecting high-grade (Gleason score ≥7) prostate cancer. They evaluated two cohorts of men including those with a subsequent positive biopsy and a subsequently negative biopsy following initial negative prostate biopsy. They then developed the EpiScore, which is a methylation intensity algorithm, which was then combined with other traditional clinical risk factors. They ultimately found that low DNA-methylation levels in prostate cancer-negative biopsies were associated with a negative predictive value of 96% for high-grade cancer[63].

The ConfirmMDx test was performed in a large sample of 605 men from a single community-based urology practice with prior negative prostate biopsy but persistently elevated PSA. Of the 605 men, the mean age was 64 years with a mean PSA of 6.8 ng/mL. In total, 308 (51%) men had a negative result and 297 (49%) men had a positive result. The repeat biopsy rate was 32.3% (96/297) for epigenetic assay-negative men and 5.8% (15/308) for epigenetic assay-positive men. The time to repeat biopsy was shorter for ConfirmMDx-positive men (median: 4 months) compared with ConfirmMDx-negative men (median: 8 months)[64]. These results highlight the ability of this test to not only influence the decision to proceed with a repeat biopsy but the time to biopsy as well.

The ultimate goal of this test is to prevent unnecessary prostate biopsies by limiting repeat biopsies in previously negative men to those who would be most at risk for having a subsequent positive result, particularly those harboring an undetected high-grade prostate cancer. The actual clinical implementation of these findings was investigated in a field observation study of 138 men from five clinical urology practices who had undergone ConfirmMDx analysis following prior negative prostate biopsy. These men had a median age of 63 years with a current median PSA level of 4.7 ng/mL. Only 6/138 (4.3%) of the men with a previously negative epigenetic assay result underwent repeat biopsy, of which all were negative for cancer. The results indicated that negative results by the ConfirmMDx assay have translated into a reduced rate of unnecessary biopsies. However, prospective clinical validation studies continue to be needed[65].

6.5 ProMark

ProMark is a protein-based quantitative immuno-fluorescence prognostic assay that quantifies the level of eight proteins. These proteins are responsible for various cellular functions including signaling and proliferation. The analyzed sample is obtained from prostate biopsy specimens[66].

This test aimed to predict the risk of harboring aggressive disease (Gleason scrore ≥4 + 3 and/or nonorgan confined diseases such as T3a, T3b, N1, or M1) in patients with Gleason scores 3 + 3 and 3 + 4 on prostate biopsy regardless of biopsy sampling variation. The test is indicated in men who have NCCN classified very-low-or low-risk disease with a life expectancy of at least 10 years. The test is scored from 0 to 1. A score of ≤0.33 is predictive of having favorable pathology in 95% of very-low- and 81.5% of low-risk patients. For scores >0.8, the risk of having nonfavorable disease is about 76.9%[67].

The basis of this biomarker test comes from the difficulty and challenges in identifying prostate cancer aggressiveness in individual samples due to variations in tumor heterogeneity, biopsy-sampling error, and variations in biopsy interpretation. Sampling variation occurs when the highest GG area of the prostate is insufficiently sampled while discordance in pathologist interpretation (as high as 30%) can further confound the diagnosis. As a result, there is a 25%–50% risk of downstaging or upstaging of prostate biopsy Gleason score following analysis of RP specimens[68]. The problem with this is that it ultimately translates into either delayed treatment for patients on AS who would have been better served with primary treatment or overtreatment of patients who may have benefited from an AS approach[69].

In 2014, Shipitsin et al. used a quantitative multiplex proteomics in situ imaging system to identify 12 biomarkers (from 160 candidates) from intact tumor epithelium that was able to accurately predict prostate cancer aggressiveness and lethal outcome. The test accounted for sampling error by incorporating biopsy samples from low and high Gleason score areas to create both high and low tumor microarrays[70].

Table 3 summarizes the various prostate tissue-based biomarkers according to molecular markers, validation and prognostic data, and FDA approval status.

7 DISCUSSION

There are several important points to consider when evaluating biomarker indication and application in clinical practice. Limitations exist including test applicability to all patient populations. Most of the cohorts utilized in the validation studies included Caucasian males from western nations. It is well established that prostate cancer incidence and mortality differ amongst races. Another significant limitation is that many of the validation studies were performed prior to widespread MRI use and therefore the utility of these tests post-MRI is yet to be understood[71].

Furthermore, head-to-head comparison of the different prostate-tissue-based genomic biomarker tests are important for comparing how the tests perform when utilized on the same patient. In 2020, Shahait et al. compared Decipher and Prolaris in a head-to-head comparison to evaluate the comparative effectiveness of each test in predicting oncological outcomes in two separate cohorts. They found a significant correlation between Decipher and Prolaris scores and the 10-year risk of BCR reported by Prolaris and the 5-year risk of metastasis reported by Decipher. While Decipher specifically performed better for predicting BCR and metastasis overall, the scores from both tests were highly correlated with respect to predicting the risk of progression following RP[72].

Alam et al. retrospectively compared Decipher, Prolaris, and Oncotype DX. They evaluated 22 patients who had at least two different genomic tests. The agreement ratio between Decipher and Prolaris, Prolaris and Oncotype Dx, and Decipher and Oncotype DX was 67%, 75%, and 50%, respectively. Prolaris was specifically noted to be most apt at confirming the recommendation of AS, while Oncotype DX was most likely to refute this recommendation[73].

Further prospective studies are needed to fully evaluate these genomic biomarkers in multiple clinical settings and with diverse patient populations to better inform clinical decision making.

8 CONCLUSION

Tissue-based biomarker discovery and development has revolutionized cancer diagnostics and therapeutic potential. In addition to helping identify actionable targets for therapy, biomarker characterization has allowed for the challenges of tumor heterogeneity and the impact of genomic variation in patient populations to be better accounted for in clinical trials. Overall, these biomarkers have proven as indispensable tools for patients and clinicians in the diagnostic and management challenges that underlie prostate cancer therapy.

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