*Article* **The Urine Biomarker PUR-4 Is Positively Associated with the Amount of Gleason 4 in Human Prostate Cancers**

**Richard Y. Ball <sup>1</sup> , Ryan Cardenas <sup>2</sup> , Mark S. Winterbone <sup>2</sup> , Marcelino Y. Hanna <sup>3</sup> , Chris Parker 4,5, Rachel Hurst <sup>2</sup> , Daniel S. Brewer 2,6 , Lauren D'Sa <sup>1</sup> , Rob Mills <sup>1</sup> , Colin S. Cooper <sup>2</sup> and Jeremy Clark 2,\***


**Abstract:** The Prostate Urine Risk (PUR) biomarker is a four-group classifier for predicting outcome in patients prior to biopsy and for men on active surveillance. The four categories correspond to the probabilities of the presence of normal tissue (PUR-1), D'Amico low-risk (PUR-2), intermediate-risk (PUR-3), and high-risk (PUR-4) prostate cancer. In the current study we investigate how the PUR-4 status is linked to Gleason grade, prostate volume, and tumor volume as assessed from biopsy (*n* = 215) and prostatectomy (*n* = 9) samples. For biopsy data PUR-4 status alone was linked to Gleason Grade group (GG) (Spearman's, *ρ* = 0.58, *p* < 0.001 trend). To assess the impact of tumor volume each GG was dichotomized into Small and Large volume cancers relative to median volume. For GG1 (Gleason Pattern 3 + 3) cancers volume had no impact on PUR-4 status. In contrast for GG2 (3 + 4) and GG3 (4 + 3) cancers PUR-4 levels increased in large volume cancers with statistical significance observed for GG2 (*p* = 0.005; Games-Howell). These data indicated that PUR-4 status is linked to the presence of Gleason Pattern 4. To test this observation tumor burden and Gleason Pattern were assessed in nine surgically removed and sectioned prostates allowing reconstruction of 3D maps. PUR-4 was not correlated with Gleason Pattern 3 amount, total tumor volume or prostate size. A strong correlation was observed between amount of Gleason Pattern 4 tumor and PUR-4 signature (*r* = 0.71, *p* = 0.034, Pearson's). These observations shed light on the biological significance of the PUR biomarker and support its use as a non-invasive means of assessing the presence of clinically significant prostate cancer.

**Keywords:** prostate; cancer; urine; PUR; PUR-4; Gleason pattern 4
