Urinary Signatures of Renal Cell Carcinoma Investigated by Peptidomic Approaches Clizia Chinello 1 * . , Marta Cazzaniga 1. , Gabriele De Sio 1 , Andrew James Smith 1 , Erica Gianazza 1 , Angelica Grasso 2 , Francesco Rocco 2 , Stefano Signorini 3 , Marco Grasso 4 , Silvano Bosari 5 , Italo Zoppis 6 , Mohammed Dakna 7 , Yuri E. M. van der Burgt 8 , Giancarlo Mauri 6 , Fulvio Magni 1 1 Department of Health Science, University of Milano-Bicocca, Monza, Italy, 2 Department of Specialistic Surgical Sciences, Urology unit, Ospedale Maggiore Policlinico Foundation, Milano, Italy, 3 Department of Laboratory Medicine, Hospital of Desio, Desio, Italy, 4 Department of Surgical Pathology, Cytology, Medical Genetics and Nephropathology, Azienda Ospedaliera San Gerardo, Monza, Italy, 5 Department of Medicine, Surgery and Dental Sciences, Pathology Unit, Ospedale Maggiore Policlinico Foundation Milano, Italy, 6 Department of Informatics, Systems and Communication, University of Milano-Bicocca, Milano, Italy, 7 Mosaiques Diagnostics GmbH, Hannover, Germany, 8 Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands Abstract Renal Cell Carcinoma (RCC) is typically asymptomatic and surgery usually increases patient’s lifespan only for early stage tumours. Moreover, solid renal masses cannot be confidently differentiated from RCC. Therefore, markers to distinguish malignant kidney tumours and for their detection are needed. Two different peptide signatures were obtained by a MALDI- TOF profiling approach based on urine pre-purification by C8 magnetic beads. One cluster of 12 signals could differentiate malignant tumours (n = 137) from benign renal masses and controls (n = 153) with sensitivity of 76% and specificity of 87% in the validation set. A second cluster of 12 signals distinguished clear cell RCC (n = 118) from controls (n = 137) with sensitivity and specificity values of 84% and 91%, respectively. Most of the peptide signals used in the two models were observed at higher abundance in patient urines and could be identified as fragments of proteins involved in tumour pathogenesis and progression. Among them: the Meprin 1a with a pro-angiogenic activity, the Probable G-protein coupled receptor 162, belonging to the GPCRs family and known to be associated with several key functions in cancer, the Osteopontin that strongly correlates to tumour stages and invasiveness, the Phosphorylase b kinase regulatory subunit alpha and the SeCreted and TransMembrane protein 1. Citation: Chinello C, Cazzaniga M, De Sio G, Smith AJ, Gianazza E, et al. (2014) Urinary Signatures of Renal Cell Carcinoma Investigated by Peptidomic Approaches. PLoS ONE 9(9): e106684. doi:10.1371/journal.pone.0106684 Editor: Ivano Eberini, Universita ` degli Studi di Milano, Italy Received June 20, 2014; Accepted July 31, 2014; Published September 9, 2014 Copyright: ß 2014 Chinello 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. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: FM was supported by grants from the Italian Ministry of University and Research: FIRB 2007 (Rete nazionale per lo studio del proteoma umano, no.RBRN07BMCT_11; http://www.ihpnet.it), Fondi Ateneo 2010-2013 (http://www.unimib.it/), and in part by the EuroKUP COST Action (BM1104) Mass Spectrometry Imaging: New Tools for Healthcare Research (http://www.maldi-msi.org/). The research leading to these results has also received funding from the European Union’s Seventh Framework Programme FP7 2007-2013 under grant agreement FP7-PEOPLE-2013-ITN-608332 (http://www.imodeckd.org/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journal’s policy and the authors of this manuscript have the following competing interests: MD is employee of Mosaiques Diagnostics GmbH. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * Email: clizia.chinello@unimib.it . These authors contributed equally to this work. Introduction Biomarkers able to characterize and predict multifactorial diseases, such as cancer, are still one of the most important targets for all the ‘‘omics’’ investigations. These clinically oriented studies have also been successfully performed in the peripheral fluids, taking advantage of non- or very low-invasive collection methods. In particular, the urinary low-molecular-weight proteome, also termed urinary peptidome [1,2], represents an important source of information for biomarker discovery. The analysis of the urinary peptidome should be most applicable to renal diseases, given that urine should contain a higher amount of molecules, including these naturally occurring polypeptides, with an altered concentration deriving directly from kidney. In particular, Renal cell carcinoma (RCC) needs markers for detection, prognosis and therapeutic targeting [3]. Whereas RCC includes an heterogeneous group of tumours with variable clinical outcomes, that range from indolent to explicitly malignant [4], the most common histological type is represented by clear cell RCC (ccRCC), and comprises approximately 60% of all renal tumours [5]. RCC is the third most frequent malignancy of the genitourinary tract accounting for about 90% of all renal malignancies and the most fatal urological cancer, causing approximately 2% of all cancer deaths [6]. It is noteworthy that this carcinoma is one of the human cancers with an increasing incidence. Currently, as RCC is typically asymptomatic, most cases are frequently detected as an incidental renal mass, imaging the abdomen for other reasons such as during the work-up of acute renal failure [5]. About 30% of RCC patients will present metastases at the time of the diagnosis, many others will develop metastasis after surgical resection and for PLOS ONE | www.plosone.org 1 September 2014 | Volume 9 | Issue 9 | e106684