A Novel Gene Signature for Molecular Diagnosis of Human Prostate Cancer by RT-qPCR Federica Rizzi1,2., Lucia Belloni1,2., Pellegrino Crafa3, Mirca Lazzaretti3, Daniel Remondini4, Stefania Ferretti5, Piero Cortellini5, Arnaldo Corti6, Saverio Bettuzzi1,2* 1 Department of Medicina Sperimentale, University of Parma, Parma, Italy, 2 Istituto Nazionale Biostrutture e Biosistemi (I.N.B.B.), Roma, Italy, 3 Department of Patologia e Medicina di laboratorio, University of Parma, Parma, Italy, 4 Dimorfipa, University of Bologna, Bologna, Italy, 5 Urology Operative Unit, Azienda Ospedaliera-Universitaria of Parma, Parma, Italy, 6 Department of Scienze Biomediche,University of Modena, Modena, Italy Abstract Background: Prostate cancer (CaP) is one of the most relevant causes of cancer death in Western Countries. Although detection of CaP at early curable stage is highly desirable, actual screening methods present limitations and new molecular approaches are needed. Gene expression analysis increases our knowledge about the biology of CaP and may render novel molecular tools, but the identification of accurate biomarkers for reliable molecular diagnosis is a real challenge. We describe here the diagnostic power of a novel 8-genes signature: ornithine decarboxylase (ODC), ornithine decarboxylase antizyme (OAZ), adenosylmethionine decarboxylase (AdoMetDC), spermidine/spermine N(1)-acetyltransferase (SSAT), histone H3 (H3), growth arrest specific gene (GAS1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Clusterin (CLU) in tumour detection/classification of human CaP. Methodology/Principal Findings: The 8-gene signature was detected by retrotranscription real-time quantitative PCR (RT- qPCR) in frozen prostate surgical specimens obtained from 41 patients diagnosed with CaP and recommended to undergo radical prostatectomy (RP). No therapy was given to patients at any time before RP. The bio-bank used for the study consisted of 66 specimens: 44 were benign-CaP paired from the same patient. Thirty-five were classified as benign and 31 as CaP after final pathological examination. Only molecular data were used for classification of specimens. The Nearest Neighbour (NN) classifier was used in order to discriminate CaP from benign tissue. Validation of final results was obtained with 10-fold crossvalidation procedure. CaP versus benign specimens were discriminated with (8065)% accuracy, (8166)% sensitivity and (7867)% specificity. The method also correctly classified 71% of patients with Gleason score,7 versus $7, an important predictor of final outcome. Conclusions/Significance: The method showed high sensitivity in a collection of specimens in which a significant portion of the total (13/31, equal to 42%) was considered CaP on the basis of having less than 15% of cancer cells. This result supports the notion of the ‘‘cancer field effect’’, in which transformed cells extend beyond morphologically evident tumour. The molecular diagnosis method here described is objective and less subjected to human error. Although further confirmations are needed, this method posses the potential to enhance conventional diagnosis. Citation: Rizzi F, Belloni L, Crafa P, Lazzaretti M, Remondini D, et al. (2008) A Novel Gene Signature for Molecular Diagnosis of Human Prostate Cancer by RT- qPCR. PLoS ONE 3(10): e3617. doi:10.1371/journal.pone.0003617 Editor: Andrew Vickers, Memorial Sloan-Kettering Cancer Center, United States of America Received June 3, 2008; Accepted October 2, 2008; Published October 31, 2008 Copyright: 2008 Rizzi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Contract grant sponsors: Genprofiler Srl, Bolzano, Italy; FIL 2005 and FIL 2006, University of Parma, Italy; Istituto Nazionale Biostrutture e Biosistemi (INBB), Roma, Italy Competing Interests: The authors have declared that no competing interests exist. * E-mail: saverio.bettuzzi@unipr.it . These authors contributed equally to this work. Introduction Prostate cancer (CaP) is believed to become the most relevant cause of cancer death in the Western Countries in the near future because its incidence increases rapidly with age. Since prognosis is generally unfavourable when disease is no longer organ-confined, detection of CaP at an early curable stage is highly desirable, but unfortunately available methods such as serum Prostate Specific Antigen (PSA) present limitations [1]. The diagnosis of CaP is conventionally obtained by saturation prostate biopsy and morphological examination of tissue sections. This method is reliable, but requires careful training. Nevertheless, intra- and inter-observer incongruities may occur. In principle, molecular diagnosis would be more objective and, hopefully, significantly less subjected to human error if obtained with reliable methods. But the ideal method should also be fast, standardized and economically convenient. Such achievement is indeed possible in theory, but results published are not completely satisfactory yet, also because they are usually based on a conventional approach with takes advantage of a single molecular predictor significantly up- or down-regulated in the cancer specimen versus benign control. A major obstacle to this goal is that the molecular events causing CaP onset and progression are still far from being completely revealed: very few well known oncogenes or tumor- suppressors have been clearly linked to prostate tumorigenesis, and for this reason CaP is still considered an elusive disease. Therefore, PLoS ONE | www.plosone.org 1 October 2008 | Volume 3 | Issue 10 | e3617