**5. Other potential biomarkers**

#### **5.1 Metabolomics**

8 Prostate Cancer – Diagnostic and Therapeutic Advances

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

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).

(Zehentner et al., 2006; Zielie et al., 2004).

**4.3 MicroRNAs** 

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 cancer aggressiveness or biochemical progress.

#### **5.2 Disseminated tumor cells**

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 presence of DTCs at the time of initial diagnostic.

Biomarkers of Aggressiveness in Prostate Cancer 11

treatment response), studies on patients at earlier stages are hampered by the low CTC counts. A recent study from Haber and collaborators shows that tumor cells obtained from the blood of cancer patients were monitored before and after surgery, most circulating cells rapidly declined shortly after surgery while others persisted months thereafter, suggesting that postoperative CTCs might derive from preestablished non prostatic sites of disease that continue to shed CTCs into the circulation (Stott et al., 2010). If confirmed, these observations support the potential application of CTC monitoring as a marker of invasive

We have here attempted to give some examples of potential DNA-based, RNA-based and protein-based markers of aggressiveness in prostate cancer. Comparisons between studies are often difficult because of some inconsistencies between study cohorts, collection methods and handling of samples. It is unlikely that a single biomarker (evaluated on conventional approach looking at a single molecular predictor significantly up- or downregulated) will provide the information requested to tell how aggressive a diagnosed prostate cancer is. New research methods (proteomics, metabolomics…) are also emerging, and high-throughput technologies will facilitate biomarker discovery. Therefore, future advances in this field will probably have to integrate proteomics, transcriptomics and multiplex approaches and identify combinations of multiple biomarkers in order to improve

The authors are supported by the Association Française d'Urologie, the Association pour la Recherche sur les Tumeurs de Prostate, the Institut National du Cancer, the Ligue Nationale contre le Cancer (Equipe labellisée), the Université Paul Sabatier de Toulouse, the Hôpitaux

Abrahamsson, P.A.; Lilja, H.; Falkmer, S.& Wadstrom, L.B. (1988). Immunohistochemical

Albadine, R.; Latour, M.; Toubaji, A.; Haffner, M.; Isaacs, W.B.; E, A.P.; Meeker, A.K.;

Ananias, H.J.; van den Heuvel, M.C.; Helfrich, W.& de Jong, I.J. (2009). Expression of the

Attard, G.; Clark, J.; Ambroisine, L.; Fisher, G.; Kovacs, G.; Flohr, P.; Berney, D.; Foster, C.S.;

Prostate, Vol.69, No.10, (Jul 1), pp.1101-1108, ISSN 1097-0045

distribution of the three predominant secretory proteins in the parenchyma of hyperplastic and neoplastic prostate glands. Prostate, Vol.12, No.1, pp.39-46, ISSN

Demarzo, A.M.; Epstein, J.I.& Netto, G.J. (2009). TMPRSS2-ERG gene fusion status in minute (minimal) prostatic adenocarcinoma. Mod Pathol, Vol.22, No.11, (Nov),

gastrin-releasing peptide receptor, the prostate stem cell antigen and the prostatespecific membrane antigen in lymph node and bone metastases of prostate cancer.

Fletcher, A.; Gerald, W.L.; Moller, H.; Reuter, V.; De Bono, J.S.; Scardino, P.; Cuzick, J.& Cooper, C.S. (2008). Duplication of the fusion of TMPRSS2 to ERG sequences

localized disease before the establishment of viable metastatic lesions.

the characterization of aggressive prostate cancers.

pp.1415-1422, ISSN 1530-0285

**6. Conclusion** 

**7. Acknowledgments** 

0270-4137

de Toulouse.

**8. References** 


Table 1. Potential biomarkers and their strategy of detection

Despite bone marrow analysis provides important information, peripheral blood studies are more acceptable in the clinical management than invasive BM aspirations. However, identification of circulating tumor cells (DTCs) require extremely sensitive analytical methods that are usually combined with enrichment procedures. Although promising results from patients with advanced stages demonstrate the value of CTCs technology (currently evaluated and validated in clinical trials as a predictor and surrogate endpoint of treatment response), studies on patients at earlier stages are hampered by the low CTC counts. A recent study from Haber and collaborators shows that tumor cells obtained from the blood of cancer patients were monitored before and after surgery, most circulating cells rapidly declined shortly after surgery while others persisted months thereafter, suggesting that postoperative CTCs might derive from preestablished non prostatic sites of disease that continue to shed CTCs into the circulation (Stott et al., 2010). If confirmed, these observations support the potential application of CTC monitoring as a marker of invasive localized disease before the establishment of viable metastatic lesions.
