The management and screening of prostate cancer (PC) is still the main problem in clinical practice. In this study, we investigated the role of aggressiveness genetic markers for PC stratification. We analyzed 201 plasma samples from PC patients and controls by digital PCR. For selection and validation, 26 formalin-fixed paraffin-embedded tissues, 12 fresh tissues, and 24 plasma samples were characterized by RNA-Seq, immunochemistry, immunofluorescence, Western blot, and extracellular-vesicles analyses. We identified three novel non-invasive biomarkers; all with an increased expression pattern in patients (PCA3: p = 0.002, S100A4: p ≤ 0.0001 and MRC2: p = 0.005). S100A4 presents the most informative AUC (area under the curve) (0.735). Combination of S100A4, MRC2, and PCA3 increases the discriminatory power between patients and controls and between different more and less aggressive stages (AUC = 0.761, p ≤ 0.0001). However, although a sensitivity of 97.47% in PCA3 and a specificity of 90.32% in S100A4 was reached, the detection signal level could be variable in some analyses owing to tumor heterogeneity. This is the first time that the role of S100A4 and MRC2 has been described in PC aggressiveness. Moreover, the combination of S100A4, MRC2, and PCA3 has never been described as a non-invasive biomarker for PC screening and aggressiveness.

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