Prostate cancer biomarkers: An update

Urologic Oncology: Seminars and Original Investigations ] (2013) ∎∎∎–∎∎∎

Seminar article

Prostate cancer biomarkers: An update Javier Romero Otero, M.D.a, Borja Garcia Gomez, M.D.a, Felix Campos Juanatey, M.D.a,b, Karim A. Touijer, M.D.c,d,* a

Hospital Universitario 12 Octubre, Madrid, Spain Hospital Universitario Marques De Valdecilla, Santander, Spain c Urology Service, Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY d Department of Urology, Weill Medical College of Cornell University, New York, NY b

Abstract Many aspects of prostate cancer diagnosis and treatment could be greatly advanced with new, effective biomarkers. Prostate-specific antigen (PSA) has multiple weaknesses as a biomarker, such as not distinguishing well between cancer and benign prostatic hyperplasia or between indolent and aggressive cancers, thus leading to overtreatment, especially unnecessary biopsies. PSA also often fails to indicate accurately which patients are responding to a given treatment. Yet PSA is the only prostate cancer biomarker routinely used by urologists. Here, we provide updated information on the most relevant of the other biomarkers currently in use or in development for prostate cancer. Recent research shows improvement over using PSA alone by comparing total PSA (tPSA) or free PSA (fPSA) with new, related markers, such as prostate cancer antigen (PCA) 3, the individual molecular forms of PSA (proPSA, benign PSA, and intact PSA), and kallikreins other than PSA. Promising results have also been seen with the use of the fusion gene TMPRSS2:ERG and with various forms of the urokinase plasminogen activation receptor. Initially, there were high hopes for early PCA, but those data were not reproducible and thus research on early PCA has been abandoned. Much work remains to be done before any of these biomarkers are fully validated and accepted. Currently, the only markers discussed in this paper with Food and Drug Administration-approved tests are PCA 3 and an isoform of proPSA, [-2]proPSA. Assays are in development for most of the other biomarkers described in this paper. While the biomarker validation process can be long and filled with obstacles, the rewards will be great—in terms of both patient care and costs to the health care system. r 2013 Elsevier Inc. All rights reserved. Keywords: Prostate cancer; Biomarkers; Prostate-specific antigen; Prostate cancer antigen 3

Introduction In Europe and the United States, prostate cancer is the most common solid neoplasm and the second leading cause of deaths due to cancer in men [1,2]. The use of prostatespecific antigen (PSA) as a prostate cancer screening tool has led to a downstaging and downgrading of the disease at the time of diagnosis and a reduction in prostate cancer mortality. However, PSA-based screening is also associated with overdiagnosis and overtreatment. The fact that PSA is synthesized by all prostate epithelial cells, whether normal, hyperplastic, or cancerous, weakens the specificity of PSA as a cancer biomarker. Elevated serum PSA levels may reflect the presence of cancer or may be caused by benign prostatic

* Corresponding author. Tel.: þ1-646-422-4486; fax: þ1-212-988-0768. E-mail address: [email protected] (K.A. Touijer).

1078-1439/$ – see front matter r 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.urolonc.2013.09.017

hyperplasia (BPH), infection, and chronic inflammation. PSA requires interpretation within the context of the given clinical scenario. Additional variation in PSA levels is introduced by the different analytical methodologies. Consequently, despite its tremendous value in clinical practice, PSA is not the ideal biomarker for prostate cancer detection and management. For this reason, countless efforts have been made to develop prostate cancer biomarkers. The National Institutes of Health define “biomarker” as a trait that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmaceutical response to a therapeutic intervention [3]. Cancer biomarkers are produced either by the tumor or by the body in response to the tumor. Various types of biomarkers can be used in the detection of prostate cancer depending on the clinical circumstances: early detection/screening, diagnosis, prognosis, prediction, therapeutic target, and evaluating a surrogate end point [4].

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In recent years, the medical literature has displayed a rapidly increasing interest in biomarkers. A number of biomarkers have subsequently been discovered and studied, but to date, only 1 biomarker is routinely used by urologists— PSA. This reflects the complex analytical and regulatory challenges for applying biomarkers in prostate cancer care. These challenges include the status of intellectual property protection, availability of standard reference materials for assays, complexity of assay formats, implementation of quality control to assure reproducibility and accuracy, sufficient market testing size to assess commercialization methods, lack of clear guidelines for good manufacturing/ laboratory practice, lack of quality control requirements for all phases of biomarker development, and cost and effort required to accumulate clinical data under appropriately designed, prospective trials. The interval required for resolution of patent issues and assay standardization and for validation, testing, and regulatory approval is also an inhibiting factor [5]. Biomarker research is generally done within the context of standard clinical care, not clinical trials, and has largely been guided by intuition and experience rather than wellstructured analyses. Thus, most biomarker findings are not reproducible. Indeed, most biomarkers that have appeared to be biomedically and statistically significant at a center are not confirmed by others [6]. In 2002, the National Cancer Institute's Early Detection Research Network developed a highly regulated process based on a 5-phase approach to systematic discovery and evaluation of biomarkers mimicking drug development, which is a highly regulated process [7]. With this rigorous framework in mind, this review describes the status of prostate cancer biomarkers currently in use or under development. A PubMed/Medline search was conducted to identify original articles from January 2000 to March 2003. The searches were limited to articles in English. The keywords included prostate cancer and biomarkers or markers. The articles with highest level of evidence or at the validation stage were selected and reviewed with the consensus of the authors of this article.

Prostate cancer antigen 3 In 1999, Bussemakers et al. [8] were the first to publish their findings regarding a new prostate cancer–related gene, DD3. Using the polymerase chain reaction (PCR) method, they saw this gene was overexpressed in prostate tumor tissue, it had low rates of expression in hyperplastic prostate tissue, and it could not be quantified in the normal tissue of many organs, including the prostate, testicles, bladder, kidney, seminal vesicles, brain, and lungs. It was named prostate cancer antigen 3 (PCA3) and corresponds to a noncoding region of the 9q2122 chromosome, the function of which is unknown. de Kok et al. [9] confirmed Bussemakers' findings, observing that the PCA3 messenger RNA (mRNA) was expressed 6 to 34 times more in the tumor tissue than in healthy tissue.

Initial development phase for PCA3 assays With these findings, Hessels et al. [10] applied the notion that following transrectal massage, prostate cells could be found in urine so as to quantify urinary PCA3 mRNA. As PSA is only slightly overexpressed in tumor cells in comparison with healthy cells, they introduced the concept of the PCA3 score, obtained by dividing PCA3 mRNA by PSA mRNA. The authors found that, for any given cutoff, the PCA3 score exhibited sensitivity and specificity rates of 67% and 83%, respectively, based on a study of 108 patients undergoing biopsy for serum PSA levels 43 ng/ml. Its superiority was confirmed in other studies using a new evolution of the test that compared urinary PCA3 with PSA in patients preselected for a prostate biopsy owing to elevated serum PSA levels [11]. In 2006, Groskopf et al. [12] demonstrated the greater stability of PCA3 at room temperature and redesigned the test using samples that were collected in a single test tube following prostate massage and later analyzed. Numerous trials were subsequently conducted based on this PCA3 test, with discrimination rates varying from 94% to 100% [13,14], superior to those reported for earlier versions of the test.

Clinical application of PCA3 The studies mentioned thus far were investigating the value of this marker to reduce unnecessary biopsies. Marks et al. [15] were the first to study the value of PCA3 in 226 patients who underwent a subsequent biopsy, demonstrating its superiority to PSA. Nevertheless, the mean PCA3 values did not discriminate between high-grade (Gleason score Z7) or low-grade (Gleason score o7) tumors. Therefore, 2 multicenter prospective trials (1 European and 1 US trial) were carried out in patients undergoing a first or second biopsy. Both studies reported a comparable area under the curve (AUC) (0.65 vs. 0.68). The European study observed a slightly greater predictive value in the second biopsy than in the first, which contrasted with the results of the American trial. Both studies concluded that by combining PCA3 with other established risk factors such as age, rectal examination, prostate volume, and percentage of fPSA, diagnostic accuracy was enhanced in multivariate regression models [14,16]. In line with these results, Ankerst et al. [17] revealed that incorporating PCA3 into the risk calculator of the Prostate Cancer Prevention Trial improved diagnostic accuracy compared with previously established risk factors. Chun et al. [18], in a sample of 809 patients, showed that adding PCA3 to the established risk factors improved the predictive values of the nomograms by 2% to 5%. In fact, these new nomograms have been externally validated and represent another tool in clinical decision making in urology. Using Chun's nomogram, a recent study avoided 21% of unnecessary biopsies at the expense of losing 6.8% of tumors [19]. Even further,

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Hansen et al. [20] found that when applying nomogramderived probability thresholds r30%, only a few cases of high-grade prostate cancer (r2%) would be missed while avoiding up to 55% of the biopsies. It is also important to consider the cost of the technique. At an Austrian center, the PCA3 test cost €243.10 compared with €32.40 for the PSA test. However, savings derived from a lower number of unnecessary biopsies would have to be factored in, as well as the costs derived from follow-up tests (fPSA and PSA following intervention) [21]. PCA3 has also been assessed as a potential screening marker in the European Randomised Study of Screening for Prostate Cancer Trial, but given that it was applied to patients who had previously been screened on the basis of PSA, its results should be interpreted with caution, and new studies should be carried out in as-yet-unscreened populations [15]. Although there are no conclusive data yet, the usefulness of PCA3 has also been evaluated in active surveillance patients [21]. The effect of medical intervention, whether invasive or noninvasive, on PCA3 was examined by Larre et al. [22] in 15 patients in whom the levels were measured before and after prostate biopsy; no significant differences were found. Another study, performed in 16 patients, detected no relationship between dutasteride and PCA3 levels [23]. In February 2012, the US Food and Drug Administration (FDA) approved the use of the Progensa test (Gen-Probe, San Diego, CA) in “patients aged 50 years or older who have had one or more previous negative prostate biopsies and for whom a new biopsy would be recommended by a urologist based on the current standards of care,” in an attempt to avoid a new biopsy or delay it if the Progensa result was negative [24]. Furthermore, earlier studies had used a cutoff score for Progensa results of 35; the FDA approval set a much more sensitive cutoff of 25 [25]. The 2012 update to the European Association of Urology guidelines for prostate cancer states that the main indication for the PCA3 test might be to determine if a patient should undergo a repeat biopsy following one that was initially negative, although it emphasizes that the procedure's cost-effectiveness is yet to be determined [26]. Table 1 summarizes the most important trials related to PCA3 in the last 10 years.

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PCA3 summary The PCA3 score, a comparison of urinary PCA3 mRNA levels to PSA mRNA levels, is a promising but so far unvalidated prostate cancer biomarker. PCA3 has proven to be superior to tPSA and the ratio of fPSA in detecting prostate cancer in patients with elevated PSA levels, given that slight, yet significant, increases in the AUC are observed with the detection of positive biopsies. The PCA3 score might be used together with PSA and other risk factors in nomograms or other methods of risk stratification in making decisions regarding whether to perform a first or successive biopsy. The most widely studied, and therefore the clearest, indication is in patients who have already undergone biopsy with a negative result. PSA isoforms PSA is a kallikrein-like serine protease produced almost exclusively by the epithelial cells of the prostate. For practical purposes, it is organ specific but not cancer specific, with circulating levels that correlate with the disruption of the prostate basal membrane epithelial cells (BPH, prostatitis, or trauma to the prostate). Thus, serum levels may be elevated in the presence of nonmalignant conditions. PSA level as an independent variable is a better predictor of cancer than suspicious findings on digital rectal examination or transrectal ultrasound [29]. PSA circulates in blood in its free form (5%35% of tPSA) or attaches to serum protease inhibitors, forming complex PSA. There are 3 molecular forms of fPSA in the serum, each contributing roughly one-third of fPSA: 33% proPSA (which has 5 isoforms), 28% benign PSA (BPSA), and 39% intact PSA (iPSA) [30]. ProPSA PSA is normally secreted from the prostatic epithelial cells as proPSA, an inactive proenzyme containing 244 amino acids. Once released into the prostate lumen, the 7-amino acid peptide is eliminated extracellularly by human

Table 1 PCA3 trials References

No. of patients

Biopsies reported/total biopsies

Percentage of PCa

AUC

Sensitivity

Specificity

PPV

NPV

Hessels et al. [10] Haese et al. [14] Marks et al. [15] Deras et al. [16] Ankerst et al. [17] Chun et al. [18] Perdonà et al. [19] Roobol et al. [27] van Gils et al. [28]

108 463 226 570 443 809 218 721 534

336/351 463/463 226/233 557/570 443/443 809/809 218/218 721/721 534/583

28 27.6 26.5 36.1 27.8 39.1 33.5 16.9 33.0

0.72 0.66 0.68 0.69 0.67 0.68 0.83 0.64 0.66

67 47 58 54 63 81 70 68 65

83 72 72 74 60 45 81 56 66

53 39 43 58 38 49 65 24 48

90 78 83 74 81 79 83 90 80

NPV ¼ negative predictive value; PPV ¼ positive predictive value.

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kallikrein enzymes hK-2 and hK-4, becoming the active or mature form of PSA with 237 amino acids. The forms with some part of the peptide yet bound to them remain as proPSA [31]. The ratio of proPSA to fPSA has proven its usefulness, when determined in the serum of patients with cancer who had tPSA levels between 2.5 and 4.0 ng/ml and between 4.0 and 10.0 ng/ml in screening programs. In a study of 119 men (88 with no cancer and 31 with cancer), at a fixed sensitivity of 75%, specificity was significantly greater for percentage of proPSA at 59% compared with percentage of fPSA at 33% (P o 0.0001), which indicated that 75% of these cancers could potentially be detected using percentage of proPSA and sparing 59% of unnecessary biopsies, whereas the use of percentage of fPSA would result in sparing only 33% of unnecessary biopsies in the 2.5 to 4.0 ng/ml tPSA range. Identification of the cancer samples was also improved by percentage of proPSA in samples with 4 to 10 ng/ml tPSA and percentage of fPSA o15%, with optimal results in the subgroup of patients with percentage of fPSA o15% [32]. Another study on 161 patients enrolled in a cancer early detection biomarker program with a percentage of fPSA less than 15% showed that the ratio of proPSA and BPSA can distinguish cancer with greater accuracy when the percentage of fPSA is very low (o15%), and may, therefore, provide better clinical utility in this lower range of percentage of fPSA [33]. Elevated proPSA to fPSA ratios have also been associated with aggressive pathologic features and decreased biochemical disease-free survival after radical prostatectomy (RP) in a retrospective revision of 555 specimens from patients who had PSA levels from 2 to 4 and 536 specimens from patients with levels from 4 to 10 ng/ml [34]. In a prospective cohort of 71 men enrolled into active surveillance for prostate cancer, serum and tissue levels of proPSA at diagnosis were associated with the need for subsequent treatment [35]. [-2]proPSA and other proPSA isoforms In proPSA, the smaller the part bound to the peptide in the leader region, the more difficult it is to activate (and the higher the percentage, owing to persistence of proPSA in tumor tissues). This makes [-2]proPSA the most stable component of proPSA in the serum [31] as [-2]proPSA is produced much more in the periphery of the prostate, particularly if it is neoplastic. There are significant differences with the transitional zone (in prostatic tissue studies), which would have implications for determining the tumor origin of the PSA elevations in the initial stages of prostate cancer [36]. Although other proPSA isoforms may be present in significant levels in serum samples, [-2]proPSA appears to be more consistently correlated with prostate cancer [31]. In men with PSA levels between 6.0 and 24.0 ng/ml, the [-2]proPSA fraction was found to be significantly higher in men with prostate cancer [30].

Prospective studies demonstrated that [-2]proPSA better enhanced discrimination between prostate cancer and benign disease compared with PSA and percentage of fPSA. Despite the fact that initially the use of percentage of [-2]proPSA to identify aggressive tumors (Gleason score 47) had not been conclusive [37,38], there is growing evidence that it correlates with unfavorable histopathology (pT3 category and Gleason score Z7) [39]. There has even been a study that correlates the percentage of [-2]proPSA/percentage of fPSA with the need for treatment in patients on active surveillance [35]. Moreover, there has been evidence that a mathematical regression model (Beckman Coulter Prostate Health Index [phi], approved by the FDA in June 2012) that combines PSA, fPSA, and [-2]proPSA provided a better overall result for discrimination in the range of 2 to 10 ng/ml PSA [40]. An automated tool using the [-2]proPSA assay with percentage of fPSA–based artificial neural network was capable of detecting prostate cancer and aggressive disease with higher accuracy than PSA or percentage of fPSA alone [41]. The 2 isoforms [-5]proPSA and [-7]proPSA have been seen to appear in different percentages in prostate cancer than they do in BPH, in the PSA range of 4 to 10 ng/ml. However, they do not enhance accuracy with respect to tPSA and percentage of fPSA [42]. In contrast, the determination of [-5, -7]proPSA in tissue surrounding the tumor in biopsies of active surveillance patients correlated with greater need for treatment [35]. BPSA or BPH-associated PSA BPSA is a degraded form or an internally cleaved form between Lys182 and Ser183 of fPSA [30] that is more often associated with BPH in prostate tissue [43]. BPSA is expressed in nodular hyperplasia limited to the transitional zone in men with BPH and can be detected in the semen, blood, and prostate tissue. Its levels correlate with obstructive voiding symptoms and show a direct association with transitional zone volume, with BPSA being a better predictor of prostate enlargement than tPSA and fPSA [43,44]. BPSA is not affected by age and is significantly higher in the presence of BPH symptoms. Adjusting fPSA level for BPSA resulted in 13% to 17% improvement in specificity over fPSA alone, while maintaining a sensitivity of 90% to 95% [45]. “Intact” PSA iPSA consists of mature PSA and proPSA which, owing to enzymatic action, has undergone structural or conformational changes that render it inactive [31]. Its concentrations initially revealed improved discrimination between BPH and prostate cancer [46]. This parameter, as a part of a multikallikrein panel, also aided in decreasing unnecessary biopsies in screening cohorts [47]. In a study, iPSA's relevance can be found in percentage of fPSA, revealing

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that high iPSA concentrations, especially lower iPSA/tPSA ratio, correlate with high stage and tumor grade in patients undergoing RP [48]. The fusion gene TMPRSS2:ERG Overview Fusion genes are the result of structural chromosomal aberrations (translocations, deletions, or inversions) made up of 2 chromosomal regions that rupture and change position, thus being able to merge into a new gene with a new function [49]. Fusion genes have been amply characterized in lymphomas, leukemias, and sarcomas, and more recently, in carcinomas such as those of the prostate, thyroid, and kidney [49]. In prostate cancer, a recurring fusion of genes has been identified in recent years—the gene TMPRSS2 (21q22.3), regulated by androgens, and ERG, from the ETS family (21q22.2), which is the most overexpressed gene in prostate cancer [50]. Currently, more than 20 different fusions have been reported implicating ERG with TMPRSS2, generally caused by an interstitial deletion at locus 21q22 and a reciprocal translocation. Other fusions of TMPRSS2 with other genes from the ETS family have also been described, although they are uncommon [51].

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specific abundance of androgen-regulated ETS gene fusions including TMPRSS2:ERG in early-onset prostate cancers, whereas elderly onset prostate cancers displayed primarily non–androgen associated structural rearrangements [55]. Other authors found no correlation between a positive test for TMPRSS2:ERG and cancer outcomes following RP [56]. TMPRSS2:ERG might also be an interesting treatment target, as BCR-Abl is imatinib's target in chronic myeloid leukemia [51]. In cell lines containing the reordered gene, decreased cell growth has been detected with histone deacetylase inhibitors and with estrogen receptor agonists [57]. In patients with castration-resistant prostate cancer, treatment with abiraterone has reduced PSA by more than 90% in carriers of this genetic fusion [58]. Some groups have combined PCA3 and TMPRSS2:ERG with the aim of increasing cancer detection and developing a urine test for prostate cancer. Hessels points out that PCA3 sensitivity for detecting prostate cancer increases from 63% to as high as 72% by including the fusion gene [53]. The combination of TMPRSS2:ERG with other markers such as alpha-methylacyl-CoA racemase, PSA, or sarcosine or with rectal examination has enhanced the predictive value of the models [59]. The limitations of TMPRSS2:ERG, beyond its doubtful prognostic value, are its low frequency in some populations (rare in the Asian population) and the difficulty of identifying a cutoff that would be applicable to all populations.

Clinical implications for TMPRSS2:ERG Other prostate cancer biomarkers Unlike PCA3, there is no test to detect this genetic reordering available for clinical use. The fusion of TMPRSS2 with ERG appears in 40% to 70% (approximately 50%) of all diagnosed prostate cancers, which, in light of the high prevalence of prostate cancer, makes this fusion the most widely reported genetic aberration in a solid tumor [52]. Notably, TMPRSS2:ERG has not been detected in biopsies of benign lesions. An assay to detect TMPRSS2:ERG would be greatly useful for men exhibiting consistently elevated PSA levels with a prior negative results on a biopsy. Similar to PCA3, TMPRSS2:ERG can be detected in the urine following a prostate massage. The detection of this fusion gene in the urine has a specificity rate of more than 90% and a positive predictive value of 94% for prostate cancer, implying that it could be a straightforward test for the presence or absence of cancer [53]. This chimeric gene can also be detected in frozen, formaldehyde-preserved, and paraffin-embedded surgical specimens and even in circulating cells. To date, there is no consensus regarding the prognostic implications of this genetic alteration, possibly because of the heterogeneity of the studies and the different techniques used to determine its presence (mainly, reverse-transcriptase PCR (RT-PCR) and in situ fluorescence hybridization) [51]. Some studies indicate that the fusion is associated with a more aggressive, higher-stage cancer phenotype, metastasis, and cancer-specific mortality [54]. A recent study found a

Urokinase plasminogen activator Urokinase plasminogen activator (uPA) can be considered a biomarker insofar as it is related to various evolutive time points in prostate cancer. The inactive precursor of serine protease, uPA, binds to a specific soluble cell surface receptor (uPA receptor [uPAR]), promoting the transformation of plasminogen into plasmin. Plasmin subsequently degrades a wide spectrum of extracellular matrix proteins by activating a cascade of proteases. Increased serum levels of different uPAR forms have been associated with poor prognosis (and above all, with distant metastases) in several cancers, including prostate and bladder [60]. A study found a trend toward increased risk of death from prostate cancer in patients with screening-detected prostate cancer who had increased values of either soluble uPAR (suPAR) form (intact or cleaved). Being in the highest quartile of either of the 2 suPAR forms was associated with a highly significant increased risk of cardiovascular death, with age-adjusted hazard ratio of 3.27 (95% CI 1.387.73) for suPAR (I–III) quartile 4 vs. quartile 1 [61]. Specific measurements of different uPAR forms in the serum improve the specificity of prostate cancer detection. Serum cleaved uPAR domain I (uPAR [I]) and uPAR (II–III)

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were higher in prostate cancer than in benign disease. In men with tPSA of 2 to 10 ng/ml, the combination of percentage of fPSA with the uPAR (I)/uPAR (I–III) ratio had a greater area under the receiver operating characteristic AUC (0.73) than did percentage of fPSA (0.68) [62]. In biopsy specimens of patients with an elevated PSA level, uPAR fragments were significant predictors of prostate cancer [63]. Immunohistochemical staining of RP specimens revealed that overexpression of uPA and its inhibitor (PAI-1) and uPAR were associated with aggressive prostate cancer recurrence [60] and poor pathologic features, including stage, Gleason score, lymphatic invasion, surgical margin status, and lymph node metastasis [64]. Elevated circulating levels of uPA and uPAR have been linked to prostate cancer stage and bone metastases [60,61,65] in patients treated with RP. Preoperative plasma uPA was a strong predictor of biochemical recurrence after surgery. Both preoperative uPA and uPAR were associated with features of aggressive biochemical recurrence such as development of distant metastasis, suggesting an association with occult metastatic disease at the time of local therapy [66]. uPAR forms are promising prognostic and predictive markers in prostate cancer. High serum levels of each of the uPAR forms (cleaved and intact) were significantly associated with short overall survival in patients with metastatic prostate cancer [67]. A minimally invasive procedure based on a novel polymeric photosensitizer, uPA-sensitive, prodrug is currently being developed. This prodrug is selectively converted to its photoactive form by uPA, which is overexpressed by prostate cancer cells. Irradiation of the activated photosensitizer exerts a tumor-selective phototoxic effect. These promising results exhibit excellent selectivity, with the potential of being used for both imaging and therapy for localized prostate cancer [68]. Human kallikreins Tissue kallikrein and kallikrein-related peptidases comprise a family of 15 highly conserved, secreted serine proteases observed in several diseases and particularly in endocrine-related human malignancies; in fact, PSA is a member of this family (KLK3). The well-documented relationship between tissue kallikrein status and the clinical outcome in patients with cancer has led to kallikreins being seen as promising diagnostic, prognostic, and treatmentresponse monitoring biomarkers for some entities including prostate cancer [69]. Human kallikrein 2 (KLK2) shares an 80% sequence homology with PSA, and both are primarily expressed in the prostate gland. Similar to PSA, serum KLK2 is present in 2 forms in the blood: bound to various protease inhibitors and the preponderance is free in the circulation. Several studies have shown that, when used in conjunction with fPSA and tPSA, serum KLK2 could improve the identification of men with prostate cancer vs. men

without cancer [70]. The ratio of KLK2 to fPSA serum levels has been reported to successfully distinguish patients with prostate cancer from those with BPH [71]. The addition of KLK2 to 3 other kallikreins (tPSA, fPSA, and iPSA) improved prediction of prostate biopsy results in men with an elevated PSA level, increasing AUC from 0.68 to 0.72 and 0.83 to 0.84 and halving the number of biopsies, missing only 3 of 40 high-grade tumors, considering a risk of prostate cancer of 20% [47]. High serum level of KLK2, together with low percentage of fPSA, is a strong predictor of poor outcome for treated patients, revealing rapid disease progression and relapse [72]. It has also been suggested that KLK2 could predict poor differentiation, extracapsular extension, and biochemical recurrence in patients treated with RP [72]. The use of RT-PCR for clinical monitoring of patients with prostate cancer and detection of disseminating prostate cancer has been under investigation for almost 10 years, but the published results are still controversial [73]. Further studies are needed to establish the real potential of this molecule in the everyday management of prostate cancer screening. For KLK11, elevated serum levels were found in patients with prostate cancer compared with healthy ones [74] and the KLK11/tPSA ratio was found to be promising for discriminating malignant disease from BPH, encouraging its clinical use to prevent unnecessary biopsies [75]. The detection of KLK2-expressing cells using a RT-PCR to reveal the presence of biologically and clinically significant occult prostate cancer metastases in histopathologically normal lymph nodes can be useful in patients with locally advanced prostate cancer, given that KLK2 is strongly associated with prostate cancer progression, failure following salvage radiation therapy, development of clinically evident metastases, and prostate cancer–specific mortality after surgery [76]. Finally, in vitro treatment of androgenindependent prostate cancer cell lines (PC3 and DU145) with broadly used chemotherapeutic agents (mitoxantrone, docetaxel, etoposide, doxorubicin, and carboplatin) alters KLK5 mRNA expression, which in the future could be used to monitor patient response to chemotherapy [77,78]. Early PCA Early prostate cancer antigen (EPCA) is a nuclear matrix protein found to have altered expression related to prostate carcinogenesis. Dhir et al. [79] were able to predict prostate cancer development after 5 years by measuring anti-EPCA antibodies in prostate biopsies with negative results. Immunohistochemical analysis has sensitivity and specificity 480% for detecting prostate cancer [79,80]. EPCA assay sensitivity for patients with prostate cancer was 92% and specificity was 94% [81]. The EPCA-2.19 and EPCA-2.22 assays were able to differentiate localized prostate cancer from metastatic disease and men with BPH from those with prostate cancer [81]. However, close examination of the methods used to

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measure EPCA-2 found major deficiencies, which included reporting values beyond the assay's detection limit and using inappropriate agents to “capture” EPCA-2 (e.g. use of undiluted serum instead of a specific antibody) [82]. In September 2009, the company that had sponsored this research for many years (Onconome) filed a lawsuit against the investigators and their institutions. In response to this lawsuit, the lead author of these publications, who had assayed EPCA-2 in the samples in the first article [81], stated that he was the only person to ever get the EPCA-2 assay to work and that it only worked for him once (never before, never since) [83]. Since then, it can no longer be considered a valid biomarker. Conclusion Prostate cancer risk spans a wide spectrum ranging from completely indolent to lethal. The introduction of PSA and its use as screening tool have contributed to early detection and reduced mortality from prostate cancer. However, it has also resulted in overdiagnosis and overtreatment. Many aspects of prostate cancer can benefit greatly from the use of biomarkers, including improving diagnostic performance, detecting aggressiveness, predicting outcome, or monitoring response to therapy. New promising biomarkers are in development. Compliance with the rigorous standards of validation and judicious use within the appropriate context will improve their usefulness in clinical practice and bring us closer to the personalized care model. References [1] Ferlay J, Steliarova-Foucher E, Lortet-Tieulent J, Rosso S, Coebergh JW, Comber H, et al. Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012. Eur J Cancer 2013;49: 1374–403. [2] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin 2013;63:11–30. [3] Ilyin SE, Belkowski SM, Plata-Salaman CR. Biomarker discovery and validation: technologies and integrative approaches. Trends Biotechnol 2004;22:411–6. [4] Shariat SF, Karam JA, Walz J, et al. Improved prediction of disease relapse after radical prostatectomy through a panel of preoperative blood-based biomarkers. Clin Cancer Res 2008;14:35–91. [5] Bensalah K, Montorsi F, Shariat SF. Challenges of cancer biomarker profiling. Eur Urol 2007;52:1601–9. [6] Check E. Proteomics and cancer: running before we can walk? Nature 2004;429:496–7. [7] Verma M, Srivastava S. New cancer biomarkers deriving from NCI early detection research. Recent Results Cancer Res 2003;163:72–84: [discussion 264-6]. [8] Bussemakers MJ, van Bokhoven A, Verhaegh GW, et al. DD3: a new prostate-specific gene, highly overexpressed in prostate cancer. Cancer Res 1999;59:5975–9. [9] de Kok JB, Verhaegh GW, Roelofs RW, et al. DD3(PCA3), a very sensitive and specific marker to detect prostate tumors. Cancer Res 2002;62:2695–8. [10] Hessels D, Klein Gunnewiek JM, van Oort I, et al. DD3(PCA3)-based molecular urine analysis for the diagnosis of prostate cancer. Eur Urol 2003;44:8–15:[discussion-6].

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