The major advantages of urine-based assays are their noninvasive character and ability to monitor prostate cancer with heterogeneous foci. Almost all urine-detectable prostate-specific markers have been recently reviewed. For this reason, we focus here on only a few promising markers which have been independently evaluated (in particular PCA3, fusion genes, TERT, AMACR, GSTP1, MMP9 and VEGF) and very recent ones (ANXA3 and sarcosine). The emphasis is also on multiplex biomarker analysis and on microarray-based analysis of fusion genes. A combination of multiple urine biomarkers may be valuable in the case of men with persistently elevated serum prostate-specific antigen and a history of negative biopsies. The emerging urine tests should help in both early diagnosis of prostate cancer and identifying aggressive tumors for radical treatment.
In developed countries, prostate cancer (CaP) is the second most frequently diagnosed cancer and the third most common cancer causing death in men.1 There are also unique problems for the treatment and prognosis of CaP owing to frequent histologic heterogeneity. In reality, this carcinoma is genetically multicentric and histologically multifocal. Both genetic and epigenetic events occur independently in intratumor foci, and hypermethylation-induced loss of gene function may be as critical as specific genetic mutations in prostate carcinogenesis. Given the poor success rate in treating advanced CaP, intervention in early stages may reduce the progression of small localized carcinoma to a large metastatic lesion, thereby reducing disease-related deaths.
The prostate-specific antigen (PSA) test has enhanced the detection and awareness of this malignancy. Serum PSA levels have been widely used for diagnostic purposes for more than 25 years but false-positive and false-negative results are still common,2 especially in the so-called ‘gray zone’ (4–10 ng ml−1), which represents a dilemma for discriminating CaP from benign prostatic hyperplasia, prostatitis and urethral manipulations which often increase PSA levels.3 Conversely, there is also a significant number of diagnosed prostate carcinomas with a PSA below 4 ng ml−1 (estimated at 20–30%) resulting in undiagnosed disease.4
Various concepts such as free to total PSA ratio (fPSA/tPSA), PSA velocity, PSA density and age-specific PSA ranges have been introduced but they do not contribute much to effective treatment of these patients and their clinical significance is still under investigation.5 Currently, most academic centers perform 12 or more transrectal guided ultrasound (TRUS) biopsies with an elevated PSA level of more than 3 ng ml−1. Despite the fact that this 12 core biopsy significantly increases the cancer detection rate, elevated PSA levels still miss prostate cancers in a significant number of patients and repeated biopsies are needed to reveal the presence of CaP after previous negative biopsy.6 Thus, there is still a great demand for new markers to improve the CaP diagnosis and prevent unnecessary prostate biopsies, especially in the serum PSA ‘gray zone’, where these days there are many newly diagnosed men.
Advantages and limitations of novel urine markers
Urine is readily available and can be used to detect either exfoliated cancer cells or secreted products. The major advantages of urine-based assays are their noninvasive character and ability to monitor CaP with heterogeneous foci (see below). Almost all urine-detectable prostate-specific markers have recently been reviewed (Table 1).7, 8 For this reason, we focus here only on the promising markers which have been independently evaluated by several groups and on the very recent ones. Subsequent chapters are devoted to the current most intensively studied markers, prostate cancer antigen 3 (PCA3) and fusion genes.
AMACR, an α-methylacyl coenzyme A racemase (also known as P504S) is involved in β-oxidation of branched-chain fatty acids and fatty acid derivates. AMACR is consistently upregulated at both the mRNA and protein levels in prostate tissue,9 and several studies have also analyzed its presence in the urine of CaP patients. Western blot analysis for AMACR was used on voided urine after TRUS and biopsy, showing a 100% sensitivity and 58% specificity for CaP detection in the group of patients with negative biopsy findings.10 In another study, the quantification of AMACR transcripts normalized to PSA transcripts in prostate secretions was predictive of CaP.11 On the other hand, AMACR transcripts normalized to PSA were not statistically significant predictors of CaP in a recent multiplex study of urine markers (see below).12
GSTP1 (glutathione S-transferase P1) belongs to a family of enzymes involved in protecting DNA from free radicals. CaP is associated with the loss of GSTP1 expression due to promoter hypermethylation. This DNA alteration appears in more than 90% of prostatic carcinoma tissues and several studies have also been done on urine samples.13, 14, 15, 16, 17 In these studies the sensitivity was found to be between 19 and 76% and specificity ranged from 56 to 100%. The lowest sensitivity was found in the study in which urine was collected without previous prostatic massage.15 When assessed, no significant association was found with either Gleason score13, 14 or tumor stage.15, 16 Two studies investigated another DNA marker, loss of heterozygosity (LOH), which is the most common deletion event in CaP.18, 19 Cussenot et al.18 assessed four locations (7q, 8p, 13q and 16q) and obtained a 73% sensitivity and 67% specificity for LOH at one or more of the locations. Including two additional locations (7q, 8p, 12p, 13q, 16q and 18q), Thuret et al.19 obtained 87% sensitivity and 44% specificity.
Matrix metalloproteinases (MMPs) have been implicated in invasion and metastasis of human malignancies. Moses et al.20 used substrate gel electrophoresis (zymography) to determine MMPs in the urine of patients with a variety of cancers. MMP9 yielded better sensitivity (64%) than MMP2 (39%) for CaP whereas specificities (84 and 98%, respectively) were calculated from controls of both sexes.20 The same group also detected several unidentified urinary gelatinase activities with molecular weights >125 kDa and recently used chromatography, zymography and mass spectrometry for their identification.21 The approximately 140, >220 and approximately 190 kDa gelatinase species were identified as MMP9/TIMP1 complex, MMP9 dimer and ADAMTS7, respectively. MMP9 dimer and MMP9 were independent predictors for distinguishing between patients with prostate and bladder cancer (P<0.001 for each). Urinary MMP and VEGF (vascular endothelial growth factor) levels were also reported as predictive markers of 1-year progression-free survival in cancer patients treated with radiation therapy.22 In regard to VEGF, urinary levels have been found to be significantly higher in CaP patients than in healthy controls23 and were also predictive of survival of hormone refractory CaP.24
Telomerase reverse transcriptase (TERT) maintains the telomeric ends of chromosomes and if telomerase is active, cancer cells may escape cell cycle arrest and replicative senescence. Several groups have measured telomerase activity with the telomeric repeat amplification protocol assay and obtained sensitivities of 58, 90 and 100% and specificities of 100, 87 and 89%, respectively.25, 26, 27 Meid et al.25 also found a significant association between Gleason score and telomerase activity. Crocitto et al.17 measured TERT mRNA expression by reverse transcription-PCR and obtained a sensitivity and specificity of 36 and 66%, respectively. In all four studies, prostatic massage was performed.
One of the most recent CaP markers, annexin A3 (ANXA3), belongs to a family of calcium and phospholipid binding proteins that are implicated in cell differentiation and migration, immunomodulation, bone formation and mineralization in CaP metastasis.28 The presence of ANXA3 in urinary exosomes29 and prostasomes28 might be the reason for its remarkable stability in urine.30 ANXA3 has been quantified by western blot in the urine samples of patients with negative digital rectal examination (DRE) findings and low total PSA (2–10 ng ml−1), which is the clinically relevant group facing the biopsy dilemma.30 Combined readouts of PSA and urinary ANXA3 gave the best results with the area under the ROC curve of 0.82 for a total PSA range of 2–6 ng ml−1, 0.83 for a total PSA range of 4–10 ng ml−1 and 0.81 in all patients. ANXA3 has an inverse relationship to cancer and, therefore, its specificity was much better than that of the PSA. The staining pattern of ANXA3 in prostatic tissue was reported to correlate with Gleason score and was able to differentiate lower and higher malignant cases.31 Moreover, staining had also apparent correlation during the whole process of prostatic transformation, ranging from benign prostatic hyperplasia via prostatic intraepithelial neoplasia (PIN) to the various stages of CaP.
Prostate cancer antigen 3
Using differential display analysis, PCA3 (DD3; differential display 3) was first described by Bussemakers et al.32 in 1999. The PCA3 gene maps to chromosome 9q21-22, a region that is not frequently affected in prostatic tumors. The PCA3 gene consists of four exons whereas exon 2 is deleted from most transcripts (present in only 5% of the transcripts) and alternative polyadenylation can occur at three different positions in exon 4. There is a high density of stop codons in all three open reading frames, and thus, PCA3 belongs to the class of noncoding RNAs whose biological role in normal and diseased prostate still remains to be elucidated. It should be noted that the prostate-specific splicing variants contain exon 4 as these have been found only in prostate cancer specimens and in LNCaP cell line.33 The diagnostic and prognostic value of PCA3 in normal, hyperplastic and malignant prostate tissues was determined by qRT-PCR and compared with TERT levels (see above).34 Interestingly, PCA3 was expressed in low levels in normal prostate but not in other normal tissues, blood or tumor samples of other origin than prostate. The median increase in mRNA expression in tumor tissues compared with nonmalignant prostate tissues was much higher for PCA3 (34-fold) than for TERT (6-fold) which is advantageous for detecting the few malignant cells shed into blood, urine, prostatic massage fluid or ejaculate.
PCA3 transcripts were also determined by reverse transcription (RT)–PCR both in tissues and urine sediments.35 The amplification products (after 35 cycles of PCR) were quantified by time-resolved fluorescence-based hybridization on streptavidin-coated microplates. Prostate tumors showed a 66-fold upregulation of PCA3 in more than 95% of cancer cases compared to benign prostate tissue. For urine samples, the sensitivity of the assay was 67%, with 8 out of 24 cancer patients having low PCA3 levels. This might be in agreement with the hypothesis that false-negative samples may represent a subgroup of prostate tumors that have less tendency to invade the prostate ductal system and thus, shed less cells into the urine.36 Of 84 men with negative biopsies (specificity 83%), 14 had high PCA3 levels and their follow-up using repeated biopsies would be of great interest. This could confirm the fact that the increase in PCA3 can precede the histological diagnosis of CaP. The same research group similarly analyzed the next 583 patients undergoing biopsy. The sensitivity for the test was 65% and the specificity 66% with this cohort of patients.37
Fradet et al.38 analyzed 517 patients undergoing biopsy at five centers and reported a sensitivity of 66% and specificity 89%. They used uPM3 assay which comprise isothermic nucleic acid sequence-based amplification39 and detection of the amplification products by real-time fluorescence using specific beacon probes.40 Despite good performance, the uPM3 was withdrawn from the market after introduction of the APTIMA assay (Gen-Probe, San Diego, CA, USA; PROGENSA for European countries) which provides several advantages: (1) The ability to use whole urine and detection of lower concentrations of RNA in clinical samples (as opposed to urine sediments used in methods discussed above); (2) The target capture technology using magnetic particles is more quantitative and user friendly than the RNA extraction methods required for RT–PCR; (3) The APTIMA PCA3 assay can be completed in <6 h, and because of its robustness and reproducibility it can be implemented in the clinical laboratory.41 Several large studies have confirmed the good performance of this assay,42, 43, 44, 45, 46 and the clinical results have recently been reviewed.47 The addition of PCA3 to the urologist's diagnostic tools will not result in a state of certainty but the diagnostic sensitivity, specificity and predictive value are incrementally improved by its inclusion.47
Gene fusions in prostate cancer
For over 30 years, genetic rearrangements have been recognized as key events in cancer development. Many hematological malignancies and sarcomas are characterized by common, recurrent chromosomal translocations that lead to expression of fusion genes or deregulation of oncogenes. In contrast, epithelial carcinomas show many nonspecific chromosomal rearrangements and until recently, recurrent translocations were not considered to play a major role.48, 49
The first prostate gene fusions, untranslated region of TMPRSS2 fused to ERG or ETV1 transcription factors, were discovered by Tomlins and colleagues in 2005.50 TMPRSS2 is a type II transmembrane serine protease (21q22.3) expressed in normal prostate epithelium and involved in many physiological and pathological processes but its exact biological function is still unknown. TMPRSS2 is strongly androgen-regulated presumably through androgen response elements in the promoter/enhancer region (5′ untranslated end) to which potential oncogenes may be transposed (with their 3′ end).51, 52 Both ERG and ETV1 belong to the ETS (E26 transformation-specific) transcription factor family. Other members of the ETS transcription factor family can also be transposed to TMPRSS2 but less frequently (ERG>ETV1>ETV4>ETV5)53 and these together with other recently described fusion genes in CaP are summarized in Table 2.
The most common oncogene transposed to TMPRSS2 is ERG (v-ETS avian erythroblastosis virus E26 oncogene homolog), also called p55 or ERG-3, which maps to chromosome 21q22.3. ERG responds to mitogenic and/or stress signals transduced by various mitogen-activated protein kinases, and modulate transcription of target genes favoring tumorigenesis. Chromosomal translocations involving ERG have been found in Ewing sarcoma, myeloid leukemia and cervical carcinoma.54 Apropos CaP, the most common variants involve TMPRSS2 exon 1 or 2 fused to ERG exons 2, 3, 4 or 555, 56 with a prevalence of the exon 4.57, 58, 59 Less common combinations include TMPRSS2 exon 4 or 5 fused to ERG exon 4 or 5 59 and TMPRSS2 exon 2 fused to inverted ERG exons 6-4.60
TMPRSS2/ERG fusion prostate cancers appear to have a more aggressive phenotype and poor prognosis. Cases with TMPRSS2/ERG rearrangement through deletion are associated with higher tumor stage, higher PSA recurrence and metastases to the pelvic lymph nodes.61, 62 Rajput et al.57 found a strong association between higher Gleason pattern and TMPRSS2/ERG gene fusion. Another study found no association with Gleason score; however, the positivity of fusion status was associated with histological patterns that have been linked to more aggressive CaP (for example, intraductal tumor spread).63 Patients with gene fusions were reported to have significantly higher rates of recurrence than those lacking TMPRSS2/ERG.64 Furthermore, Demichelis et al.58 found statistically significant association between TMPRSS2/ERG and CaP-specific death in a Swedish population with up to 22 years of clinical follow-up without curative treatment (watchful waiting cohort).
However, controversial results have been reported in several studies for correlations between gene fusions and prognosis. Several important positive prognosticators (longer recurrence-free survival, well- and moderately differentiated stages, lower pathological stage and negative surgical margins) were surprisingly associated with TMPRSS2/ERG fusions.65 Similar findings have been reported in another study with a clear tendency for fusion-positive tumors to be associated with lower Gleason grade and better survival than fusion-negative tumors.66 No significant association was found between TMPRSS2/ERG status and tumor stage, Gleason grade or recurrence-free survival in another study.55 The reason of such controversial results could be small sample size in almost all studies listed above. Another reason might be insufficient follow-up and the fact that patient cases and controls were not derived from the same source making it difficult to interpret correlations with specific tumor parameters.
It is noteworthy that TMPRSS2/ERG fusion was not sufficient for transformation from PIN to cancer state in the absence of secondary molecular lesions (for example, loss of NKX3-1 or PTEN) and vice versa.67 Also, ERG overexpression itself without fusion partners markedly increased cell invasion in benign prostate cells but did not result in transformation; however, TMPRSS2/ERG fusion was necessary to mediate the PIN to CaP transition. Interestingly, a novel ER (estrogen receptor)-dependent regulation pathway was found for TMPRSS2/ERG-positive prostate cancers as an alternative mechanism by which prostate cancers might develop androgen independence from an initial androgen-dependent state.68 This should be through ERα stimulation of the TMPRSS2 promoter in castration-resistant prostate cancer, which leads to CaP progression, metastasis and more aggressive phenotype. In contrast, ERβ may function as a tumor suppressor through negative regulation of TMPRSS2/ERG expression as shown in vitro by using of ERβ agonist.
A recent study made on a cohort of prebiopsy and preradical prostatectomy patients showed that the detection of TMPRSS2/ERG fusion transcripts in the urine was feasible.69 Urine RNA was analyzed by qRT-PCR after preamplification (whole transcriptome amplification) and then break-apart fluorescent in situ hybridization (FISH) was used to validate the presence or absence of the TMPRSS2/ERG gene rearrangement in the CaP tissue. Indeed, patients with high levels of ERG and detectable levels of TMPRSS2/ERG in their urine were positive for ERG rearrangement. With regard to the association of gene fusion status and patient outcome, the urine test with subsequent confirmatory FISH might contribute to differentiating indolent from aggressive forms of CaP.
Multiplex analysis of urine markers and future directions
Multiplex or combined model of urine biomarker analysis has several advantages in CaP detection. Most importantly, it does not ignore the heterogeneity of CaP and can detect prostate cancer more accurately than single marker tests.70, 71 Recent studies have characterized the clonality and heterogeneity of TMPRSS2/ERG fusion in multifocal CaP.57, 72 Both interfocal heterogeneity and intrafocal homogeneity for TMPRSS2/ERG fusion have been found in multifocal CaP in the radical prostatectomy specimens. Current biopsy strategies may miss heterogenous tumor foci, and urine-based assay would have a great advantage because the cells from multiple cancerous foci of whole prostate could be released and collected.69
Laxman et al.12 reported that a multiplex panel of urine transcripts outperforms PCA3 transcript and serum PSA alone in detecting CaP. Expression of seven putative prostate cancer biomarkers was measured (PCA3, PSA, GOLPH2, SPINK1, AMACR, TMPRSS2/ERG, TFF3) by qRT-PCR in urine samples.12 They showed that increased GOLPH2 (Golgi phosphoprotein 2), SPINK1 (serine peptidase inhibitor, Kasal type 1), PCA3 transcript expression and TMPRSS2/ERG fusion status were significant predictors of CaP (sensitivity 66% and specificity 76%).12 Improved sensitivity to detect CaP has also been reported for combined detection of TMPRSS2/ERG and PCA3 transcripts in urine on a sample of patients with serum PSA ⩾3 ng ml−1 and/or an abnormal DRE.73 Separate sensitivities for detecting TMPRSS2/ERG fusion and PCA3 transcripts in the urine were 37 and 62%, respectively, but by combining both markers the sensitivity increased to 73%.
Simple and sensitive exon array-based assays were recently invented for simultaneous detection of multiple fusion genes in specimens with only a minor population of tumor cells.74, 75 Current methods seem to have a lot of drawbacks compared to the novel array-based assays. For example, the most commonly used FISH50, 57, 61 has relatively low resolution and, therefore, cannot accurately determine different fusion variants in highly heterogeneous samples with small percentages of tumor cells. Quantitative RT-PCR and sequencing are relatively easy to perform;56, 59, 69, 76 however, multiple sets of primers/probes are required for assessing multiple potential fusion variants. Array CGH (comparative genome hybridization) has a high resolution but often fails when there is normal cell contamination.61, 77
Recently, sarcosine (N-methyl derivative of the amino-acid glycine) has been identified as a differentially expressed metabolite that is greatly elevated during CaP progression to metastasis and, importantly, can be detected noninvasively in urine.78 It is worth noting that a relationship has been found among the sarcosine pathway, androgen signaling and ETS family of gene fusions. Sarcosine levels were directly increased by androgens in VCaP (ERG-positive) and LNCaP (ETV1-positive) cell lines indicating that components of the sarcosine pathway may have potential as biomarkers of CaP progression along with AR and ETS gene fusions and could also serve as a new target for therapeutic interventions.
The urine-based assays can monitor prostate cancer with heterogeneous foci and provides a noninvasive alternative to multiple biopsies. Even if urine assays cannot detect cancers which do not shed tumor cells into urine, it still deserves considerable attention. The combination of multiple urine biomarkers could be of special value in men who have persistently elevated serum PSA and a history of negative biopsies. The emerging biomarkers (ANXA3, sarcosine, gene fusions, PCA3 and others) should help in both early diagnosis of CaP and identifying aggressive tumors for radical treatment.
Conflict of interest
The authors declare no conflict of interest.
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This work was supported by grants NS 9940-4 from the Czech Ministry of Health and MSM 6198959216 from the Czech Ministry of Education. T Jamaspishvili was also supported by GACR 303/09/H048 from the Grant Agency of the Czech Republic. While the manuscript was being reviewed, important details on PCA3 have been drawn to our attention.106 Clarke et al. have recently identified 4 new transcription start sites, 4 polyadenylation sites and 2 new differentially spliced exons in an extended form of PCA3. The expression of the two novel exons, exon 2a and 2b, which were highly enriched in CaP and metastases, can add a further degree of sensitivity for the detection of CaP.
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Jamaspishvili, T., Kral, M., Khomeriki, I. et al. Urine markers in monitoring for prostate cancer. Prostate Cancer Prostatic Dis 13, 12–19 (2010). https://doi.org/10.1038/pcan.2009.31
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