Background: The presence of prostate cancer (PCa) Gleason pattern 4 or 5 on biopsy is a major contraindication for active surveillance (AS). Limitations of anatomopathological morphological analysis, can severely compromise the accuracy of PCa risk stratification. In this study, expression of recurrent oncogenes (MYC and ERG), tumor suppressors (PTEN and p53) and a marker of proliferation (Ki67) were evaluated as biomarkers in order to predict whether histologically indolent Gleason pattern 3 co-occurs with unsampled higher grade disease warranting further biopsy, which may in turn inform more intensive treatment. Methods: This cross-sectional study involved 37 patients diagnosed with localized PCa and very low risk who underwent radical prostatectomy. The patients were divided into two groups according to the final pathology: Group 1, Gleason score 6 (ISUP 1, n = 17) and Group 2, Gleason score 7 (ISUP 2 or 3, n = 20). The expression of proteins p53, Ki67, ERG, MYC and PTEN in FFPE samples of neoplastic and adjacent benign prostatic tissue were evaluated by digital image immunohistochemistry. Results: The expression of ERG, Ki67, and MYC was significantly higher in neoplastic tissue when compared to benign in both groups, while expression of PTEN was significantly lower. Strong p53 staining, which is enriched for gain-of-function missense alleles, did not present a significant statistical difference. When the Gleason 3 and Gleason 4 areas of group 2 were compared, no difference was observed in the expression of any of the markers studied. In the univariate analysis, comparing the absolute expression of the biomarkers in Gleason 3 PCa samples from both groups, Ki67 was the only independent predictor of adjacent Gleason 4 (ISUP 2 or 3). In the multivariate analysis, including the relative expression of the biomarkers in the Gleason 3 PCa of the two groups, age and PSA; it was observed that only PTEN (OR = 1, CI = 1.03-1.31) presented as an independent factor of adjacent Gleason 4 (ISUP 2 or 3). Conclusions: An increase in absolute Ki67 expression and loss PTEN protein expression, associated with age and PSA, were predictors of adjacent Gleason pattern 4.