**4. RNA biomarkers**

#### **4.1 PCA3**

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

#### **4.2 AMACR**

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

Biomarkers of Aggressiveness in Prostate Cancer 9

Thus, dysregulated expression of EZH2 may be involved in the progression of prostate cancer, and miR101 might represent a marker that distinguishes indolent prostate cancer

Metabolite profiling or metabolomics, the analysis of endogenous metabolites in a biological system was recently suggested to be a promising approach to identify novel metabolites or their changes (Fredolini et al., 2010). In practice, however, analysis of the metabolome is complex because of the large range of detectable metabolites. By screening 110 samples from men's urine and blood and 42 tissue samples, Chinnaiyan and collaborators recently identified 1,126 metabolites. They identified 87 that distinguish normal prostate from prostate cancer, then narrowed down the list to 6 whose levels were higher in samples linked to localized prostate cancer and higher still in metastatic disease (Sreekumar et al., 2009). Sarcosine, an *N*-methyl derivative of the amino acid glycine, was identified as a differential metabolite that was highly increased during prostate cancer progression to metastasis. Surprisingly, the authors also provided evidence using cell cultures for a functional role of sarcosine in promoting invasive properties in these cells, whereas lowering the level of the enzyme producing sarcosine reduces invasiveness (Sreekumar et al., 2009). The potential role of urinary was reevaluated in another study, which showed that sarcosine in urine after digital rectal examination fails as a marker in prostate cancer detection and identification of aggressive tumors (Jentzmik et al., 2010). In addition to this work, the same group showed no correlation with sarcosine level in tissues and tumor stage, tumor grade or biochemical recurrence in 92 samples obtained after radical prostatectomy (Jentzmik et al., 2011). Although the lack of metastatic tissue samples was a limitation, this study establishes that sarcosine measurement in prostate tissue is not suitable to predict

The shedding of tumor cells into the circulation is a necessary condition for metastasis dissemination and the clinical relevance of the detection of disseminated tumors cells (DTCs) in bone marrow (the most prominent metastatic site in prostate and breast cancer) or in peripheral blood of patients free of apparent metastasis in under investigation. So far, only large breast cancer studies have confirmed the independent prognostic value of the bone marrow status (Berg et al., 2007). Recent studies have demonstrated an association between DTCs in bone marrow at diagnosis of nonmetastastic prostate cancer (Berg et al., 2007; Kollermann et al., 2008). Although a DTC-positive bone marrow status was associated with grading and increased risk of metastasis, the study by Berg et al. on 266 patients did not find a correlation of DTC detection and survival (Berg et al., 2007). In contrast, Köllermann et al. demonstrated the prognostic relevance of DTCs in bone marrow patients with clinically localized prostate cancer submitted to neo-adjuvant hormonal therapy followed by radical prostatectomy and a median follow-up of 44 months (Kollermann et al., 2008). This study is the first one on a large series of patients with sufficient long follow-up to clearly demonstrate an adverse prognostic effect of the

from those at risk of lethal progression

cancer aggressiveness or biochemical progress.

presence of DTCs at the time of initial diagnostic.

**5.2 Disseminated tumor cells** 

**5. Other potential biomarkers** 

**5.1 Metabolomics** 

the protein level (immunohistochemistry and western-blot) in metastatic prostate cancer compared to localized prostate cancer (Kuefer et al., 2002; Rubin et al., 2004). An association between low AMACR protein expression at diagnosis and an increased risk of biochemical recurrence and fatal prostate cancer was reported in patients diagnosed with a localized prostate cancer who underwent radical prostatectomy or not (Rubin et al., 2005). Recent findings from the same group confirmed that down-regulation of AMACR expression is associated with poorer outcomes in a cohort of 920 men diagnosed with prostate cancer. However the lack of statistical significance suggests that tumor AMACR expression at diagnosis is not a useful prognostic biomarker for lethal disease after treatment (Barry et al., 2011). Although AMACR protein can been detected in urine by western-blot, its concentration is low in serum, making the development of a serum test difficult (Rogers et al., 2004). Circulating concentrations of AMACR mRNA in urine or serum quantified by RT-PCR have been found elevated in patients but these pilot studies are limited to small series (Zehentner et al., 2006; Zielie et al., 2004).

#### **4.3 MicroRNAs**

MicroRNAs are small RNAs found to regulate mRNA function by modulating both mRNA stability and the translation of mRNA into protein. Their expression is commonly altered in solid tumors and multiple microRNAs have been shown to have oncogenic properties or act like tumor suppressor genes. Besides their therapeutic potential, microRNAs hold unique characteristics that herald them as ideal tumor markers including their stability and ease of detection (Heneghan et al., 2010). Despite the large body of work that has been published to date, only limited information is available regarding the expression levels of specific microRNAs in relation to the aggressiveness of prostate cancer. Taking advantage of the stability of tumor-derived microRNAs in circulating blood, Mitchell and co-workers found a remarkably higher level of miR-141 (46-fold increase) in a patients with metastatic prostate cancer compared to healthy control men (Mitchell et al., 2008). The first evidence of a possible prognostic relevance of microRNAs in prostate cancer was obtained from a study examining the tissue expression of 40 patients undergoing prostatectomy. The increased expression of miR-135b and miR-194 was associated with biochemical recurrence within 2 years of surgery (Tong et al., 2009). Another study, conducted on matched tumor and adjacent normal tissues obtained from 76 patients, found that high expression of miR-96 was associated with cancer recurrence after radical prostatectomy, and that prognostic information was confirmed by an independent tumor sample set from 79 pateints (Schaefer et al., 2010). The miR-221 expression is also progressively reduced in aggressive prostate cancer and metastasis and predicts clinical recurrence in patients (n=92) undergoing radical prostatectomy (Spahn et al., 2010). More recently, miR-143 and miR-145 were identified as being associated with bone metastasis of prostate cancer and involved in the regulation of epithelialmesenchymal transition (Peng et al., 2011). Interestingly, the loss of miR-101 expression during cancer progression in human tumors has been associated with overexpression of histone methytransferase EZH2 (enhancer of zeste homolog 2) (Varambally et al., 2008). Amounts of both EZH2 mRNA and EZH2 protein are increased in metastatic prostate cancer; in addition, clinically localized prostate cancers that express higher concentrations of EZH2 show a poorer prognosis (Varambally et al., 2002). In cancer cell lines, the expression and function of EZH2 are inhibited by miR-101 (Varambally et al., 2008). Thus, dysregulated expression of EZH2 may be involved in the progression of prostate cancer, and miR101 might represent a marker that distinguishes indolent prostate cancer from those at risk of lethal progression
