Biomarkers for Prostate Cancer Diagnosis from Genetic Perspectives

Introduction

Prostate cancers (PC) are frequent types of cancer in men. In 2019, in the United States alone,  there were about  174,650  new cases reported which resulted in  31,620 deaths (1). Some PC patients may stay asymptomatic and die from unrelated causes. This can be the result of the older age that leads to slower cell division and tumor growth (2). Improved consideration of the genetic and molecular pathways of PC can indicate the reason why some tumors present as a clinically silent disease, contrary to some others which are life-threatening. The change of prostate epithelial cells to tumor formation and prostate cancer can be the consequence of intricate chains of both genetic, epigenetic, and environmental issues (3).

Some risk factors like age, heritage, and having a positive history of prostate malignancies can cause a timely alarm of PC which results in overtreatment, expenses, and changing the patient’s life quality. The U.S. Preventive Services Task Force (USPSTF) suggested in contradiction to the PSA testing in 2012 that unfortunately resulted in the increased incidence of metastatic PC (4, 5). 

Prostate cancer can rarely be observed in men ≤40 years and its risk rises as patients aged. Just about ten percent of PC patients are detected in men less than sixty years and signify as early-onset PCa.  Records from the Surveillance, Epidemiology and End Results (SEER) Database indicate the early-onset growing PC (6). More than age, the ancestry of PC can be a determining risk factor and it is shown that PC risk is dramatically greater in blacks, intermediate in whites, and low in native Japanese (7). More than the age and origin of the PC  patients, having a positive family history of PC  can increase the cancer occurrence, because PC  is highly heritable (8). About 5-10% of PC patients are supposed to be mainly triggered by high-risk inherited genetic aspects or PC  predisposition genes. The meta-analysis of thirty-three epidemiologic studies indicates that the risk is more for men whose brothers have cancer more than for the men with affected fathers (9). The PC  hazard enlarged when first-degree relatives (FDR) were identified with PC at the age less than sixty-five years (10).

Prostate Cancer Diagnosis

PC exact diagnosis and sufficient staging have an important role in patients' medical care and treatment. PC conventionally is detected by digital rectal examination (DRE), prostate-specific antigen (PSA), and tracked by transrectal ultrasound (TRUS) guided biopsy. PC can be very heterogeneous cancer varies from insignificant, indolent, low-grade to big, aggressive, and life-threatening tumors.

Prostate-Specific Antigen (PSA)is a34kDa protein with serine protease or Kallikerin Related Peptidase-3 (KLK3) functioning which is coding by the gene, on chromosome 19. Its biological role is the hydrolysis of semenogeline-1, producing velocity of seminal ejaculate to assist spermatozoa mobility. Its half-life is about two days. The three/forth of circulating PSA is linked to α-1 anti-chymotrypsin and α-2 macroglobulin that can be metabolized through the liver. The residual one/fourth percent of PSA is free and is excreted into the urine. It is detectable in extraordinary amounts in seminal fluid and its detection as prostate cancer serum biomarker can happen alongside forensic analysis of seminal fluid. The age-specific reference range is based on the National Institute for Health and Care Excellence (NICE) is >2.0 for 40-49 years, >3.0 for 50-59, >4.0 for 60-69, and >5.0 for age more than 70.

PSA Densityis the serum PSA level per ml (PSA/ml serum) of prostate tissue. PSA levels are more in men who have the bigger-sized glands of the prostate. The PSA concentration is occasionally considered for the size management of large prostate in BPH (benign prostate hyperplasia). The prostate gland size is measured through transurethral ultrasound (TRUS) or computed tomography (CT) scans. The cut-off for PSA density is 0.15ngml^-1 that more than can be an indicator of prostate cancer.

PSA doubling time and PSA velocity are PSA alterations over time. PSA velocity is reported as ng/ml/year and a predictor factor. By way of illustration, a PSA of 2ng/ml and a PSA velocity of 0.5 ng/ml/year can predict to have a PSA of 2.5ng/ml in 12 months. PSA doubling time is defined as the months needed for PSA levels to double.

In cases with steady PSA levels (estimated changes from 6 to 6.1ng/ml over a year), doubling time is not reliable. So, these patients are normally shown as the “doubling time>10 years” or similar.

Digital Rectal Examination is the common prostate testing of inserting the lubricated, gloved index finger of a physician into the rectum to detect rectal cancer. DRE is an initial method to detect inflammation, abnormal growth, or cancer of the prostate and diagnose nerve difficulties specified by a decline of the usual tone of the muscles of the rectal sphincter.

Despite several new suggesting diagnostic examinations, TRUS prostate biopsy is still the gold standard test.  However, due to the wrong needle placing in the exact tumor location TRUS biopsies have false-negative results of about 15%–46% (11).  To solve false-negative biopsy results, there are suggestions of random biopsies from the prostate consisting of a sequence of 6 samples spread out in the same way which is called random 6-score biopsies (12). This can increase the chances of cancer diagnosis in men with high PSA levels and decrease false negatives. Doctors at NYU Langone's Perlmutter Cancer Center developed the national standard for a 12-core prostate biopsy which increases the samples from 6 to 12 with samples from the far edges of the gland. This method can reduce the likelihood of missed cancer significantly (13). The imaging algorithm in patients with elevated PSA is presented in figure 1.

A novel blood testing has been suggested for accurate diagnosis of aggressive PCa, based on the Queen Mary University of London reports. There is hope that the newly developed liquid biopsy components like circulating tumor cells (CTCs) would improve the biopsy results or even take the place of traditional biopsy samples. Total PSA (tPSA) is the basic biological test for cancer diagnosis. Unfortunately, PSA is an organ-specific antigen, so elevated PSA amount is seen in benign pathologies (BPH), prostatitis, and PC (14). The original PC Prevention Trial (PCPT) PC Risk Calculator (PCPTRC) posted in 2006, that if PSA more than 4.0ng/ml or if an atypical DRE was distinguished, a biopsy was recommended (15).

Figure 1. Imaging algorithm in patients with increased PSA based on Brandy’s book

In 2014, the updated PCPTRC 2.0 was suggested with the extra potential of discrimination between low-grade (Gleason grade<7) from high-grade prostate cancer (16). To increase specificity the age-adjusted PSA suggested cut off 2.5, 3.5, 4.5, and 6.5ng/ml in men in their 40s, 50s, 60s, and 70s, respectively (17). The ratio fPSA/tPSA is considered on the occasions of having regular DRE and PSA between 4 and 10ng/mL. The inferior percentage of less than 15%  can be seen in men with cancer (18). In a recent publication by Ping T. et al,  it is indicated to PSA (4.0 to 10.0ng/ml) can experiencing early prostate biopsy and fPSA/tPSA increase diagnostic accuracy, contrary to the  PSA alone in patients more than sixty years old (19). A new method to expand the medical presentation of PSA is to pool blood testing results of tPSA, fPSA, and [-2] pro PSA by a calculated formula named the Prostate Health Index (Phi). The Phi test exactly practices the formula ([-2] pro PSA/fPSA × tPSA) to recover as the complementary diagnostic biomarker when the serum PSA level is about  2–10ng/mL (20). The Gleason Score is a grading system to define the violence of cancer (21, 22). 
The Gleason Score varies from 1-5 and explains the degree of biopsy similarity to normal tissue or abnormal tissue. The PC is mostly heterogeneous and is composed of tumor cells with various grades. So, two grades are allocated to individuals, the first one is known to explain the cells that make the major part of the tumor and the second one specifies the cells of the next prevalent region.  For example, the Gleason Score 3+4=7 indicates that the tumor is mostly in grade 3 and then the possible tumor is grade 4, collectively resulting in the Total Gleason Score. The typical Gleason Scores vary from 6 to10. Scores ≤ 6 define cancer cells that appear like normal cells and propose that the cancer is probable to enlarge gradually. The score=7 shows the middle risk for aggressive prostate cancer, and scores of 8 ≤ indicated quick tumor spread; these tumors are often specified as poorly differentiated or high grade. 

Multiparametric-magnetic resonance imaging (mp-MRI) has recently revealed favorable outcomes in diagnosis, risk stratification, tumor locus, and staging of PCa (23). The systematic TRUS biopsy has the problem of underestimation and results in inaccurate risk stratification and wrong therapeutic options. So, the US and the Canadian Task Force on Preventive Health Care, just have suggested that the risks of PSA testing can be more than its profits (24). The mp-MRI combines the morphological calculation of T2-weighted imaging (T2WI) with diffusion-weighted imaging (DWI), dynamic contrast-enhanced (DCE) perfusion imaging, and spectroscopic imaging (MRSI) (25, 26). Especially T2WI and DWI have exposed important promise in cancer recognition, localization, and prostate tumor staging  (27, 28). In figure 2 a summary of PCa common diagnosis strategies is presented.

Figure 2. Common clinical testing method of prostate cancer

Genes and molecular biomarkers for prostate cancer diagnosis

There is a developing interest to find molecular biomarkers as the predictable clinical and pathologic factors to accurate prostate cancer diagnosis and personalized cancer treatment. Nevertheless, a combination of biomarkers into the standard clinical practice needs robust analytical validity supports. Several molecular biomarkers ranging from DNA mutation, DNA Methylation, and mRNA are considered for both PCa diagnosis and prognosis.

 PCA3 mRNA is a long noncoding RNA that can have a role in the prostate carcinogenesis pathway. Some long non-coding RNAs (lncRNA) are described as the important ones in PCa including Prostate-specific transcript 1 non-protein-coding (PCGEM1), PCa Associated Transcript 1 (PCAT-1), and the PC gene 3 (PCA3). The PCA3 gene, firstly named Differential Display Code 3. The coding gene of PCA3 is positioned on chromosome 9q21-22 and is composed of 4 exons and 3 introns. There is different polyadenylation at the locus of exon 4 (4a, 4b, and 4c) resulting in three transcripts with different lengths. The new genomic structure of progression PCA3 gene insertion in the intron of other genes like  PRUNE2 or BMCC1PCA3 regulates their expression by double-stranded RNA (dsRNA) formation (29).  The quantity of the mRNA PCA3 is calculated by the quantitative real-time polymerase chain reaction (qRT-PCR) technique after extracting it from urine.

The PCA3 test gained the Conformité européenne in 2006 and was accepted by the FDA in 2012. The  PCA3 test was officially confirmed by the FDA as the prostate cancer diagnostic tool in determining re-biopsy is essential or in the follow-up of prostate cancer patients in active surveillance strategy (30). Based on the published result of a meta-analysis, the general sensitivity, specificity, and area under curve (AUC) values of PCA3 mRNA were equal to 0.63, 0.88, and 0.82, respectively (31).

The Oncotype DX® is a panel of several gene expressions through RT-PCR that was recognized for fixed paraffin-embedded (FPE) diagnostic prostate needle biopsies about  1 mm (32). The Oncotype DX® PC assay dealings expression of twelve genes involving molecular pathways and five algorithmically shared genes based on the Genomic Prostate Score (GPS).  The Oncotype DX PC Assay indicates single biological pathways with an identified role in prostate tumorigenesis: the androgen pathway (Zinc-alpha-2-glycoprotein (AZGP1), Kallikrein Related Peptidase 2 (KLK2), Steroid 5 Alpha-Reductase 2 (SRD5A2), and Family With Sequence Similarity 13 Member C (FAM13C), cellular organization (Filamin-C (FLNC), Gelsolin (GSN), β-Tropomyosin (TPM2), and Glutathione S-transferase Mu 2 (GSTM2), proliferation Xenopus kinesin-like protein 2 (TPX2), stromal response (Biglycan (BGN), Collagen Type I Alpha 1 Chain (COL1A1), and Secreted Frizzled Related Protein 4 (SFRP4)) (33). Reference gene standardization is considered for pre-analytical and analytical contradiction as well as a grant for variable RNA inputs and is predictable clinical/pathologic elements (34). The Oncotype DX^®  has been valid from tissue biopsy and enhances medical parameters like National Comprehensive Cancer Network (NCCN) scores (33, 35).

Decipher^® Prostate Cancer Test (GenomeDx Biosciences, San Diego, CA) is according to the more than twenty gene expression panel which offerings precise understanding in calculating clinical metastasis (0.75–0.83) and cancer-specific mortality (0.78). It is based on a microarray technique including 1.4 million probes complementary to target genes that detect both coding and non-coding RNA expression levels (36). These genomic marks have been advanced, protected, and authenticated based on the  Genomic Classifier data (37). The control of postoperative radiotherapy (adjuvant vs. salvage) can be shown regarding Decipher scores that can calculate the risk of tumor metastasis and recurrence in patients treated with radical prostatectomy (38). This test is the first self-determining sign of tumor metastasis in patients with biochemical recurrence after surgery that can consider in the individualized medication of prostate cancer.

ConfirmMDxfor Prostate Cancer (MDxHealth, Irvine, CA) complexes epigenetic analysis that processes DNA methylation of Glutathione S-Transferase Pi 1 (GSTP1), Adenomatous polyposis coli (APC), and Ras Association Domain Family Member 1 (RASSF1). It is an epigenetic assay to the exact diagnosis of true-negative biopsy (39). The national comprehensive cancer network (NCCN) guidelines suggested ConfirmMDx because several institutional review observations wanted this panel to be added to the repeated biopsy setting. After all, it adds value in men with a raised PSA and previous negative biopsy results (Table 1) (40). The MATLOC (Methylation Analysis to Locate Occult Cancer) confirmation established the cost-effective value of the ConfirmMDx test (41). The actual power of the ConfirmMDx test is in its negative predictive value of more than 90%.

Table 1. Substitutes to immediate biopsy can expand the specificity of PSA testing. The 2018 NCCN Guidelines mention that clinicians study biomarkers and mpMRI before performing a biopsy

PSA: Prostate Specific Antigens; fPSA: free PSA; DLX: Distal-less Homeobox 1; HOXC6: Homeobox C6; GSTP1: glutathione S-transferase pi gene; RASSF1: Ras association domain-containing protein 1

The Prostate Core Mitomic Test™57(PCMT, Mitomics, CO, USA) is a test based on several mitochondrial DNA (mtDNA) deletions and states molecular modification of malignant tumor cells in normal-appearing tissue (42). It is the usual cancer diagnostic tool in men in the first world. Since 2011, the PCMT counts a 3.4-kb mtDNA genetic deletion toughly connected to the PC, and in New Jersey about fifty urology practices retrieving pathology services of the PCMT successfully (43).

TMPRSS2-ERG fusion gene can happen in 40% - 80% of PCa in humans. The v-ets erythroblastosis virus E26 oncogene homolog 1 (ERG) genes over expressions have roles in prostate cancer mediated by the androgen receptor signaling pathway. TMPRSS2-ERG is a fusion between the transmembrane protease serine 2 (TMPRSS2) genes and the ERG gene which is linked to clinically substantial cancer (44). A combination of biomarker assays can expand prognostic precision paralleled to the procedure of separate markers (Figure 3). Calculation of post-DRE urine TMPRSS2-ERG, in grouping with urine PCA3, boosted the value of serum PSA level for expecting cancer risk (45).

Figure 3. Schematic presentation of TMPRSS2-ERG fusion

PTEN gene deletion is related to the higher Gleason grade, risk of tumor progression, and recurrence after therapy (46). PTEN is a tumor suppressor gene whose genomic aberrations are mostly common in PC patients and this gene deletion or mutation is recognized in about 20% of primary tumor samples at radical prostatectomy and in almost 50% of castration-resistant tumors (47). For patients with high-grade prostatic intraepithelial neoplasia (HGPIN) or an atypical diagnosis, PTEN assay is a practical method to discriminate between nonaggressive HGPIN or an atypical diagnosis in men at greater risk of PC.

ProMark is an advanced prognostic test established exactly for PC for biopsy Gleason Score of 3+3 or 3+4  automated image recognition technology to tell the risk of aggressive PC (48). The ProMark scores between 1 and 100. A personalized score specifies the aggressiveness of the PC to define the correct treatment strategy. It is involved in the National Comprehensive Cancer Network (NCCN) Clinical Care Guidelines (49).

4K score is a four-kallikrein panel including kallikrein-related peptidase 2 (hK2), intact PSA, fPSA, and tPSA. The 4Kscore blood test precisely identifies men with aggressive PC before prostate biopsy with or without DRE information (50). The 4Kscore Test is a pre-biopsy blood test that includes four prostate protein biomarkers and clinical information to control a man's risk for high-grade, aggressive (Gleason ≥7) PCa (50). 

Circulating tumor cells (CTCs) are tumor cells that are released from solid tumors and are circulating in the bloodstream. CTCs are the main component of “Liquid Biopsy”  indicating the analysis of tumor cells and tumor cell products such as cell-free nucleic acids (51). Very recently, circulating tumor cells (CTCs) have been studied in PC. It is suggested that CTC testing act as the highly accurate new blood test for PCa to avoid invasive biopsies (52). More CTCs numbers in the blood of CRPC patients can be an indicator of worse outcomes. Checking the gene expression profile of each CTCs and its markers can improve the knowledge of PC (53). CTCs can be useful even for predicting treatment efficacy and survival outcomes. However, CTC detection techniques with upgraded sensitivity are under investigation.

Conclusions

The PC diagnosis is commonly done by PSA, DRE, TRUS, and mpMRI to determine the Gleason score of PC. Several molecular biomarkers can improve the diagnosis and solve the problem of the false-negative result of the biopsy. These biomarkers are ConfirmMDx, Prostate Core Mitomic Test (PCMT), TMPRSS2-ERG, The PTEN gene, ProMark, 4K score, and CTCs that help the clinician to decide in the re-biopsy step.

Authors’  contributions

FKH wrote the original draft, SMKA is mainly designed and supervised the project, AS and MHKH  were responsible for data curation, and SSH reviewed and edit the manuscript. All authors reviewed and approved the final version of the manuscript.

Acknowledgments

Special thanks to the Department of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Conflict of interest

The authors declare that there is no conflict of interest.

Funding

the authors received no financial support for this research.

Ethical Statements

Not applicable

Data availability

Data will be provided by the corresponding author on request.

Abbreviations

CTCs            Circulating Tumor Cells

DRE             Digital rectal exam

ERG             ETS-related gene

fPSA/tPSA   Free PSA to total PSA

HGPIN         High-grade prostatic intraepithelial neoplasia

mp-MRI       Multiparametric-magnetic resonance imaging

PC                Prostate cancers

PCMT          Prostate Core Mitomic Test

PSA             Prostate-specific antigen

TMPRSS2   Transmembrane Serine Protease 2

TRUS          Transrectal ultrasound

USPSTF      U.S. Preventive Services Task Force