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The aim of this study was to assess the relationship between pre-prostatectomy urinary Engrailed-2 (EN2), a transcription factor secreted by prostate cancer cells, with tumour volume and pathological characteristics in resected prostate specimens. First pass urine samples (10 ml) without prior prostatic massage were collected and stored at –80°C. EN2 levels were measured using an enzyme-linked immunoabsorbent assay. Tumour volume in the prostatectomy specimens was determined histologically. 57 men undergoing RP in one urological cancer network were evaluated. EN2 was detected in 85% of RP patients. EN2 correlated with tumour volume (but not total prostatic volume) in a linear regression analysis, with increasing pathological T stage and margin positivity. Using three “cutoff levels” of tumour volume (0.5 ml, 1.3 ml and 2.5 ml) to define “significant disease”, men with “significant disease” had markedly higher levels of urinary EN2 (p < 0.001 for each cut off level). Levels of urinary EN2 may be useful in predicting tumour volume in men with prostate cancer by potentially identifying men with small volume “insignificant” disease. This study justifies a larger multicentre evaluation of urinary EN2 levels as a biomarker of PC significance using cancer volume, pathological and PSA criteria.