Targeted proteomics in urinary extracellular vesicles identifies biomarkers for diagnosis and prognosis of prostate cancer

// Tamara Sequeiros 1 , Marina Rigau 1 , Cristina Chiva 2, 3 , Melania Montes 1 , Iolanda Garcia-Grau 1 , Marta Garcia 1 , Sherley Diaz 4 , Ana Celma 5 , Irene Bijnsdorp 6 , Alex Campos 8 , Primiano Di Mauro 9 , Salvador Borros 9 , Jaume Reventos 10, 11 , Andreas Doll 1 , Rosanna Paciucci 1 , Michiel Pegtel 7 , Ines de Torres 1, 4 , Eduard Sabido 2, 3 , Juan Morote 1, 5 , Mireia Olivan 1 1 Group of Biomedical Research in Urology, Vall d’Hebron Research Institute (VHIR) and Universitat Autonoma de Barcelona (UAB), Barcelona, Spain 2 Proteomics Unit, Centre de Regulacio Genomica (CRG), Barcelona, Spain 3 Proteomics Unit, Universitat Pompeu Fabra, Barcelona, Spain 4 Department of Pathology, Vall d'Hebron University Hospital, Barcelona, Spain 5 Department of Urology, Vall d'Hebron University Hospital, Barcelona, Spain 6 Department of Urology, VU University Medical Center, Amsterdam, The Netherlands 7 Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands 8 Sanford-Burnham Medical Research Institute, La Jolla, California, USA 9 Sagetis-Biotech; Grup d'Enginyeria de Materials (GEMAT) Institut Quimic de Sarria, Barcelona, Spain 10 Departement of Basic Science, International University of Catalonia, Barcelona, Spain 11 IDIBELL-Bellvitge Biomedical Research Institute, Barcelona, Spain Correspondence to: Mireia Olivan, email: mireia.olivan@vhir.org Keywords: prostate cancer, biomarkers, urine, extracellular vesicles, diagnosis Received: September 22, 2016     Accepted: November 07, 2016     Published: November 26, 2016 ABSTRACT Rapid and reliable diagnosis of prostate cancer (PCa) is highly desirable as current used methods lack specificity. In addition, identification of PCa biomarkers that can classify patients into high- and low-risk groups for disease progression at early stage will improve treatment decision-making. Here, we describe a set of protein-combination panels in urinary extracellular vesicles (EVs), defined by targeted proteomics and immunoblotting techniques that improve early non-invasive detection and stratification of PCa patients.We report a two-protein combination in urinary EVs that classifies benign and PCa patients (ADSV-TGM4), and a combination of five proteins able to significantly distinguish between high- and low-grade PCa patients (CD63-GLPK5-SPHM-PSA-PAPP). Proteins composing the panels were validated by immunohistochemistry assays in tissue microarrays (TMAs) confirming a strong link between the urinary EVs proteome and alterations in PCa tissues. Moreover, ADSV and TGM4 abundance yielded a high diagnostic potential in tissue and promising TGM4 prognostic power. These results suggest that the proteins identified in urinary EVs distinguishing high- and low grade PCa are a reflection of histological changes that may be a consequence of their functional involvement in PCa development. In conclusion, our study resulted in the identification of protein-combination panels present in urinary EVs that exhibit high sensitivity and specificity for PCa detection and patient stratification. Moreover, our study highlights the potential of targeted proteomic approaches–such as selected reaction monitoring (SRM)–as diagnostic assay for liquid biopsies via urinary EVs to improve diagnosis and prognosis of suspected PCa patients.