**2. Protein biomarkers**

#### **2.1 Prostate Stem Cell Antigen (PSCA)**

PSCA (Prostate Stem Cell Antigen) is a membrane glycoprotein with 30% homology to stem cell antigen type 2 (SCA-2), an immature lymphocyte cell surface marker. Like SCA-2, PSCA is attached to the membrane by a GPI anchor which can be cleaved by a phospholipase. Because of its homology with SCA-2, PSCA was named inaccurately since it is not a marker for stem cells nor is it uniquely expressed in the prostate (Saeki et al., 2010). Initially, PSCA was identified as a tumor antigen overexpressed in the prostate (Reiter et al., 1998), and subsequent studies have revealed that it is also up-regulated in other cancers including bladder and pancreas. PSCA has been proposed as a promising tumor marker of diagnostic and prognosis, as well as a potential therapeutic target for patients with metastatic prostate cancer. PSCA expression indeed increases with high gleason score, advanced stage and bone metastasis (Gu et al., 2000; Han et al., 2004; Lam et al., 2005; Zhigang & Wenlv, 2004). The levels of PSCA are also amplified in the prostate intraepithelial neoplasia (PIN) lesions that

Biomarkers of Aggressiveness in Prostate Cancer 5

in patients undergoing radical prostatectomy, however only 46 blood samples were examined (Paul et al., 2005). ELISA used for initial serum EPCA measurement in 112 men with isolated high-grade prostatic intraepithelial neoplasia (HGPIN), showed a significantly higher serum ECPA level in isolated HGPIN patients with subsequent cancer than those without cancer (Zhao & Zeng, 2010). Pretreatment serum EPCA levels were also determined with an ELISA in 77 patients with clinically localized prostate cancer who underwent radical prostatectomy and 51 patients with locally advanced or metastatic disease who received primary androgen deprivation therapy, and were correlated with clinicopathological variables and disease progression. Patients with locally advanced and metastatic prostate cancer had significantly higher serum EPCA level than those with clinically localized disease. These data suggest that EPCA level correlates significantly with the poor prognosis, showing prediction potential for prostate cancer progression (Zhao et al., 2011). Unrelated to the original ECPA, the nuclear protein ECPA-2 was recently described (Leman et al., 2007). Carried out on 385 blood samples, the ECPA-2 ELISA was able to differentiate between men with and without prostate cancer with 92% specificity and 94% sensitivity, whereas the specificity of PSA was only 65% in the same population (Leman et al., 2007). More importantly, in contrast to PSA, ECPA-2 is able to discriminate men with non-organconfined prostate cancer from those with organ-confined diseases. Although additional validation studies are needed to show the performance of ECPA-2 in a more representative population, these results lend support to the development of a more accurate blood-based

A wealth of reports suggest a key role for the urokinase-type plasminogen activator and its receptor (uPA/uPAR) in invasion and metastatic dissemination (Duffy, 2004). uPA is is a member of the serine protease family synthetized and secreted as a pro-enzyme, whose activation is markedly accelerated upon binding with high affinity to specific membranebound or soluble cell surface uPAR. Once activated, the uPA/uPAR system efficiently converts plasminogen into plasmin, a protease which then modulates extracellular matrix degradation, tumor cell invasion and growth factor activation (Duffy, 2004). In prostate cancer, overexpression of uPA and uPAR (transcripts and proteins) have been reported in tumor tissues suggesting that both proteins could be associated with tumor progression. Immunohistochemical studies have shown an incremental increase in uPA/uPAR expression from benign epithelium to primary organ-confined prostate cancer, to disease extending beyond the prostate capsule, and to bone metastases (Cozzi et al., 2006; Gavrilov et al., 2001; Pulukuri et al., 2007; Usher et al., 2005). In addition, overexpression of uPA and its inhibitor PAI-1 are associated with aggressive prostate cancer recurrence in men treated with radical prostatectomy (Gupta et al., 2009). Elevated circulating levels of uPA and/or uPAR have been associated with advanced prostate cancer and bone metastases (Hienert et al., 1988a; Hienert et al., 1988b; Miyake et al., 1999). Shariat et al. have recently reported that plasma levels of uPA and uPAR are not only higher in men with prostate cancer than in healthy controls, but decrease significantly after prostatectomy (suggesting that direct local production by malignant cells significantly contributes to increased uPA and uPAR circulating levels of these markers in patients), then increase with disease progression (Shariat et al., 2007). Of note, higher preoperative uPA and uPAR were both significantly associated with shorter progression PSA doubling times, failure to respond to salvage local

assay to identify aggressive prostate cancer.

**2.4 uPA/uPAR** 

subsequently progressed to cancer compared to those that did not progress (Zhigang & Wenlu, 2007). PSCA might also represent a useful marker for metastasis detection as almost 95% of lymph nodes and bone samples with metastasis have been found positive for PSCA expression (Ananias et al., 2009). It was originally observed that PSCA mRNA bearing cells circulating in peripheral blood were identified at higher rates in prostate cancer patients with extraprostatic disease (Hara et al., 2002). The presence of *PSCA* transcripts in the peripheral blood has found to be a significant predictor of biochemical recurrence after prostatectomy in high-risk prostate cancer (Joung et al., 2010). Of note, PSCA has also been investigated as a potential target for tumor-targeted immunotherapy and for suppression by anti-PSCA antibody in animals. Further evaluation of PSCA as a clinical prostate cancer marker of aggressiveness needs to be validated.

#### **2.2 Prostate Secretory Protein 94 (PSP94)**

The ELISA dosage of β-microseminoprotein (MSP) ou PSP94 (Prostate Secretory Protein of 94 amino acids) – one of three predominant proteins secreted by the human prostate gland (Lilja & Abrahamsson, 1988) – could be potentially interesting. In a case-control study of 1,212 men with no previous history of prostate cancer, Nam et al. found that patients with low PSP94 levels had a high probability for having prostate cancer diagnosed at biopsy (Nam et al., 2006). The authors also suggested that those cancers that maintain PSP94 expression tend to be well differentiated and less aggressive, as reported in previous immunohistochemistry studies (Abrahamsson et al., 1988). In serum, PSP94 can be complexed with PSPBP (PSP-binding protein), a glycoprotein of 50 kDa sharing significant identity with the CRISP (cystein-rich seccretory protein) family of proteins (Reeves et al., 2005). In a study comprising 185 patients, it was showed that PSPBP is negatively associated with recurrence after radical prostatectomy (Reeves et al., 2006). On the contrary, PSP94 immunohistochemistry performed on 59 radical prostatectomy specimens was associated with worsened survival outcomes (Girvan et al., 2005). Another immunohistochemistry study conducted on 945 patients found a high expression of PSP94 in tumor cells patients to be associated with a longer time to postprostatectomy biochemical recurrence (Bjartell et al., 2007) in agreement with serum studies. Further studies will be needed to determine whether PSP94 and its binding protein represent novel prognostic factors in the clinic.

However, it is interesting to note that genome-wide association studies identified several SNPs in a region on chromosome 10 that harbors the *PSP94* gene (Eeles et al., 2008; Thomas et al., 2008). Its location and the strength of the association raises the possibility that this SNP may be causally related to disease risk, but resequencing and further analyses will be needed clarify the functional basis of this association.

#### **2.3 Early Prostate Cancer Antigens ECPA and ECPA-2**

ECPA and ECPA-2 (Early Prostate Cancer Antigens) are nuclear matrix proteins those alterations are commonly associated with prostate cancer (Getzenberg et al., 1991; Lakshmanan et al., 1998). Initially, immunohistochemical studies on men with negative biopsies who were ultimately found to have prostate cancer could identify individuals as much as 5 years earlier than the current diagnostic (Dhir et al., 2004). ECPA positivity was hypothesized to be an early event in disease progression, as there was no correlation between ECPA staining and Gleason grade or pT stage (Uetsuki et al., 2005). In 2005, an ECPA-based ELISA showed 92% sensitivity and 94% specificity in prostate cancer detection in patients undergoing radical prostatectomy, however only 46 blood samples were examined (Paul et al., 2005). ELISA used for initial serum EPCA measurement in 112 men with isolated high-grade prostatic intraepithelial neoplasia (HGPIN), showed a significantly higher serum ECPA level in isolated HGPIN patients with subsequent cancer than those without cancer (Zhao & Zeng, 2010). Pretreatment serum EPCA levels were also determined with an ELISA in 77 patients with clinically localized prostate cancer who underwent radical prostatectomy and 51 patients with locally advanced or metastatic disease who received primary androgen deprivation therapy, and were correlated with clinicopathological variables and disease progression. Patients with locally advanced and metastatic prostate cancer had significantly higher serum EPCA level than those with clinically localized disease. These data suggest that EPCA level correlates significantly with the poor prognosis, showing prediction potential for prostate cancer progression (Zhao et al., 2011). Unrelated to the original ECPA, the nuclear protein ECPA-2 was recently described (Leman et al., 2007). Carried out on 385 blood samples, the ECPA-2 ELISA was able to differentiate between men with and without prostate cancer with 92% specificity and 94% sensitivity, whereas the specificity of PSA was only 65% in the same population (Leman et al., 2007). More importantly, in contrast to PSA, ECPA-2 is able to discriminate men with non-organconfined prostate cancer from those with organ-confined diseases. Although additional validation studies are needed to show the performance of ECPA-2 in a more representative population, these results lend support to the development of a more accurate blood-based assay to identify aggressive prostate cancer.

#### **2.4 uPA/uPAR**

4 Prostate Cancer – Diagnostic and Therapeutic Advances

subsequently progressed to cancer compared to those that did not progress (Zhigang & Wenlu, 2007). PSCA might also represent a useful marker for metastasis detection as almost 95% of lymph nodes and bone samples with metastasis have been found positive for PSCA expression (Ananias et al., 2009). It was originally observed that PSCA mRNA bearing cells circulating in peripheral blood were identified at higher rates in prostate cancer patients with extraprostatic disease (Hara et al., 2002). The presence of *PSCA* transcripts in the peripheral blood has found to be a significant predictor of biochemical recurrence after prostatectomy in high-risk prostate cancer (Joung et al., 2010). Of note, PSCA has also been investigated as a potential target for tumor-targeted immunotherapy and for suppression by anti-PSCA antibody in animals. Further evaluation of PSCA as a clinical prostate cancer

The ELISA dosage of β-microseminoprotein (MSP) ou PSP94 (Prostate Secretory Protein of 94 amino acids) – one of three predominant proteins secreted by the human prostate gland (Lilja & Abrahamsson, 1988) – could be potentially interesting. In a case-control study of 1,212 men with no previous history of prostate cancer, Nam et al. found that patients with low PSP94 levels had a high probability for having prostate cancer diagnosed at biopsy (Nam et al., 2006). The authors also suggested that those cancers that maintain PSP94 expression tend to be well differentiated and less aggressive, as reported in previous immunohistochemistry studies (Abrahamsson et al., 1988). In serum, PSP94 can be complexed with PSPBP (PSP-binding protein), a glycoprotein of 50 kDa sharing significant identity with the CRISP (cystein-rich seccretory protein) family of proteins (Reeves et al., 2005). In a study comprising 185 patients, it was showed that PSPBP is negatively associated with recurrence after radical prostatectomy (Reeves et al., 2006). On the contrary, PSP94 immunohistochemistry performed on 59 radical prostatectomy specimens was associated with worsened survival outcomes (Girvan et al., 2005). Another immunohistochemistry study conducted on 945 patients found a high expression of PSP94 in tumor cells patients to be associated with a longer time to postprostatectomy biochemical recurrence (Bjartell et al., 2007) in agreement with serum studies. Further studies will be needed to determine whether

PSP94 and its binding protein represent novel prognostic factors in the clinic.

needed clarify the functional basis of this association.

**2.3 Early Prostate Cancer Antigens ECPA and ECPA-2** 

However, it is interesting to note that genome-wide association studies identified several SNPs in a region on chromosome 10 that harbors the *PSP94* gene (Eeles et al., 2008; Thomas et al., 2008). Its location and the strength of the association raises the possibility that this SNP may be causally related to disease risk, but resequencing and further analyses will be

ECPA and ECPA-2 (Early Prostate Cancer Antigens) are nuclear matrix proteins those alterations are commonly associated with prostate cancer (Getzenberg et al., 1991; Lakshmanan et al., 1998). Initially, immunohistochemical studies on men with negative biopsies who were ultimately found to have prostate cancer could identify individuals as much as 5 years earlier than the current diagnostic (Dhir et al., 2004). ECPA positivity was hypothesized to be an early event in disease progression, as there was no correlation between ECPA staining and Gleason grade or pT stage (Uetsuki et al., 2005). In 2005, an ECPA-based ELISA showed 92% sensitivity and 94% specificity in prostate cancer detection

marker of aggressiveness needs to be validated.

**2.2 Prostate Secretory Protein 94 (PSP94)** 

A wealth of reports suggest a key role for the urokinase-type plasminogen activator and its receptor (uPA/uPAR) in invasion and metastatic dissemination (Duffy, 2004). uPA is is a member of the serine protease family synthetized and secreted as a pro-enzyme, whose activation is markedly accelerated upon binding with high affinity to specific membranebound or soluble cell surface uPAR. Once activated, the uPA/uPAR system efficiently converts plasminogen into plasmin, a protease which then modulates extracellular matrix degradation, tumor cell invasion and growth factor activation (Duffy, 2004). In prostate cancer, overexpression of uPA and uPAR (transcripts and proteins) have been reported in tumor tissues suggesting that both proteins could be associated with tumor progression. Immunohistochemical studies have shown an incremental increase in uPA/uPAR expression from benign epithelium to primary organ-confined prostate cancer, to disease extending beyond the prostate capsule, and to bone metastases (Cozzi et al., 2006; Gavrilov et al., 2001; Pulukuri et al., 2007; Usher et al., 2005). In addition, overexpression of uPA and its inhibitor PAI-1 are associated with aggressive prostate cancer recurrence in men treated with radical prostatectomy (Gupta et al., 2009). Elevated circulating levels of uPA and/or uPAR have been associated with advanced prostate cancer and bone metastases (Hienert et al., 1988a; Hienert et al., 1988b; Miyake et al., 1999). Shariat et al. have recently reported that plasma levels of uPA and uPAR are not only higher in men with prostate cancer than in healthy controls, but decrease significantly after prostatectomy (suggesting that direct local production by malignant cells significantly contributes to increased uPA and uPAR circulating levels of these markers in patients), then increase with disease progression (Shariat et al., 2007). Of note, higher preoperative uPA and uPAR were both significantly associated with shorter progression PSA doubling times, failure to respond to salvage local

Biomarkers of Aggressiveness in Prostate Cancer 7

or lower Gleason scores (Fine et al., 2010; Gopalan et al., 2009). Taken together, it seems like the TMPRSS2:ERG fusion gene is an early event related to development of prostate cancer rather than a marker for progressive disease. Of note, the TMPRSSE:ERG fusion has potential for noninvasive prognosis of prostate cancer. Although RNA-based urinary tests demonstrate in general a high specificity and sensitivity to detect prostate cancer, no significant relationship was found between the presence of fusion transcripts and Gleason

The loss of heterozygosity (LOH) is a frequent genetic alteration in prostate cancer, in particular on chromosome arms 7q, 8p, 10q, 12p, 13q, 16q, 17q and 18q (Dong, 2006). Studies on chromosomal deletions of 8p22 by fluorescence in situ hybridization technique revealed 8p22 deletion to be the strongest parameter to predict disease progression in patients undergoing surgery (Matsuyama et al., 2001). If some LOH have been shown to be associated with early stages of prostate cancer (Lu & Hano, 2008), others seem to indicate the presence of tumor suppressor genes whose inactivation is correlated with aggressive and metastatic tumors (Dong et al., 2000; Kibel et al., 2000; Matsuyama et al., 2007). A recent study reported the development of a noninvasive method to detect early stages of prostate cancer using LOH analysis of 7q31, 8p22, 12p13, 13q14, 16q23.2 and 18q21. Indeed LOH could be found in cells from urine obtained by prostatic massage (Thuret et al., 2005). In patients who underwent radical prostatectomy, LOH was confirmed from the prostatic tissue with a concordance of 86%. This noninvasive approach warrants further investigation

PCA3 (formerly known as DD3) is a non-coding RNA very prostate specific (Bussemakers et al., 1999; de Kok et al., 2002). PCA3 mRNA levels can be measured in the urine specimens and several studies have shown that the PCA3 score is superior to serum PSA for predicting biopsy outcome (Groskopf et al., 2006; Haese et al., 2008; van Gils et al., 2007). The relationship between PCA3 score and parameters of cancer aggressiveness has also been studied and differ. Some studies report a positive relationship between PCA3 scores and parameters of more serious disease (Nakanishi et al., 2008; Whitman et al., 2008), while other studies could not find such a relationship (Hessels et al., 2010). Further studies are requested

α-methylacyl-CoA racemase (AMACR) is a catalyst in the peroximal beta-oxidation of branched chain fatty acids found in dietary sources (Wanders et al., 2001), such as red meat and dairy products, the consumption of which has been associated with increased prostate cancer risks (Hsing & Chokkalingam, 2006). AMACR has been identified as a potential biomarker based on its overexpression in localized prostate cancer as compared to benign prostate epithelium (Luo et al., 2002; Rhodes et al., 2002; Rubin et al., 2002). In fact, immunostaining for AMACR is commonly performed in prostate biopsies to help distinguish benign from malignant tissue. Of note, AMACR expression was found consistently lower both at the transcriptional (cDNA expression arrays and RT-PCR) and at

to evaluate the potential of PCA3 testing as prognostic test for prostate cancer.

score or clinical stage (Hessels et al., 2007; Rice et al., 2010).

to bring pronostic information on prostate cancer aggressiveness.

**3.3 Loss of heterozygosity** 

**4. RNA biomarkers** 

**4.1 PCA3** 

**4.2 AMACR** 

radiation therapy, and /or development of distant metastases (Shariat et al., 2007). Larger studies are needed to validate the promising role of uPA and uPAR as biomarkers of aggressive prostate cancer.
