detection rate, and prostate biopsies are more and more often becoming saturation biopsies [45]. Saturation biopsy can be considered for patients at risk of cancer who are willing to accept the side effects, but it is known that clinically insignificant cancers can be detected [46]. On the other hand, the same rationale might also explain the opposite result. In fact, the link between morphological lesions (i.e. HG-PIN) and clinical CaP is still a matter of debate [47,48]. Also PIN represents a ‘‘field effect’’ with a potential for cancer progression, but may be not directly involved in or may not lead to the development of invasive prostate cancer [49]. Therefore we might hypothesize that certain changes in morphology might not compulsory be associated to clinically relevant cell transformation. Thus, specimens classified as benign by the molecular tool, but cancer instead by the pathologist because of altered morphology (5/ 31, Table 1, bold italic), might actually be different morphological entities or clinically indolent lesions, possibly undergoing regression because surrounded by reacting tissue. If so, this material would be not significant from the clinical point of view, but clearly distinguishable on a molecular basis from aggressive disease, although we have no definitive evidence of this at the moment. Clusterin and prostate cancer CLU was cloned, sequenced and identified as the major up- regulated gene during massive induction of apoptosis and prostate regression caused by androgen depletion [7] or administration of Finasteride [50] and vitamin D analogues [51,52]. The glycosy- lated, extracellular form of CLU is produced at high basal level by prostate cells and secreted in the prostate fluid, although its possible role in reproduction is still puzzling the researchers [53]. Authors have proposed that CLU might be over-expressed in cells surviving apoptosis, and not in cells doomed to die. Thus, a cytoprotective role for CLU has been proposed [54]. The debate on this issue is still wide open. It has been recently shown that different protein forms can be originated from the same gene by still unknown mechanisms [8]. TGF-beta and X-ray treatment induce a truncated form of CLU that localizes to the nucleus [55– 58]. It is believed that different forms of CLU have different roles in human cells [8], also depending by their sub-cellular localization. Structural information concerning such protein forms are still scarce. Data concerning the possible involvement of CLU in transformation and tumour growth are still unclear or contradictory in the literature. The REST relative expression analysis demonstrated (data not shown) that the down-regulation of CLU in CaP specimens is statistically significant (p = 0.023). This is consistent with our previous results [3,13]. Although the specific issue of whether CLU is up- or down-regulated in CaP is still controversial [3,13,15,59– 61], confirmatory data supporting our hypothesis that CLU is not only down-regulated in CaP, but also in the most of cancers, can be easily retrieved from the Oncomine web site that collects data from more than 20,000 independent microarray experiments (http://www.oncomine.org). We have previously suggested that CLU might be a potential tumor-suppressor gene [51] acting through its nuclear form nCLU [57,63,64]. Potential prognostic value of the method We previously found that our informative genes have prognostic value [3,4]. It is known that Gleason score is the best single predictor of CaP prognosis [65], but prediction of outcome in the 6–7 score range, comprising the most of clinical cases, is not satisfactory [65]. We had 28/41 of patients in these conditions in our bio-bank (Table 1). Unfortunately this analysis, as done before [4], requires definitive outcome data on the whole cohort of Gene Signature patient, and thus a minimum of a 5-year follow-up study will be necessary as previously done [4]. We are strongly encouraged to pursue this possibility because our informative genes have been already found of prognostic value not only in human CaP [4], but also in the TRAMP mice model. These animals were treated with Green Tea Catechins (GTCs) for inhibition of CaP progression [19]. In this experimental model, our gene signature was capable to efficiently discriminate CaP tissue specimens of TRAMP mice responding to GTCs treatment from those not-responding, therefore suggesting that the biological behaviour of CaP might be successfully investigated also in humans by the same approach. In Table 2 it is also shown that the combination of 7 genes (H3, GAS1, H3, SSAT, CLU, AdoMetDC, OAZ, ODC) correctly classified 84% of cancer specimens with regard to age of the patient, 71% with regard to RP Gleason score and 42% with regard to PSA. Misclassification with regard to PSA was actually expected, because PSA is not a prostate tumour but, more properly, a prostate tissue specific marker. As a matter of fact, also PSA cut-off values and CaP detection rates are still a matter of debate [1]. Instead, we got a very good performance on classification of age and Gleason score, both widely used for clinical management of patients, with Gleason score being the best predictor [65]. Also these results strongly suggest that our approach is detecting important biological events occurring during prostate cell transformation and cancer progression. For the above reasons it will be fundamental to continue this study by collecting clinical follow-up data, with a particular focus on misclassified cases, for re-classification of patients as a function of final outcome and possible validation of the potential prognostic power of the method. Final remarks In conclusion, the RT-qPCR gene profiling method based on the gene signature described here appear to be an appropriate and reliable tool for molecular diagnosis of presence/absence of CaP not subjected to intra- and inter-observer error. This test could be of help to enhance diagnosis and particularly in those cases in which it is necessary to resolve possible uncertainties. Our approach, based on detection of a multiparametric expression signature, yielded the best performance at detection of CaP ever published with single predictors as far as we know. The method has potential prognostic value, because we previously demonstrat- ed that the expression level of these genes correlate with the final outcome of CaP patients. Future research should be conducted to obtain the gene signature using less invasive means than a biopsy. Materials and Methods Prostate Tissue Specimens Collection/Handling and Pathological Classification: Ethical approval for this work was granted by local Hospital Ethical Committee (Azienda Ospedaliera-Universitaria di Parma) and informed written consent was obtained from patients involved. Prostate surgical specimens were obtained from patients diagnosed with CaP by biopsy and recommended to undergo RP. No therapy was given to patients at any time before RP. Tissue specimens were excised out from the prostate gland immediately after surgery and quickly frozen in dry ice as previously described [3]. Two tissue specimens were obtained from the same prostate gland: one from the (supposed) cancerous portion of the gland, according to prostate mapping by biopsy. The second one from the (supposed) benign tissue taken very far away (i.e. in different areas of the prostate, ideally in the opposite sextant) from the neoplastic lesion. Both specimens were quickly frozen with dry ice to preserve RNA integrity, then embedded in OCT and frozen- PLoS ONE | www.plosone.org 6 October 2008 | Volume 3 | Issue 10 | e3617