Introduction: Prostate cancer (PCa) is predominantly indolent disease with a small fraction of patients undergoing metastatic progression. Therefore, it is critical to identify non-invasive diagnostic markers for the early detection of an aggressive PCa subtype. We hypothesized that non-invasive pre-digital rectal examination (pre-DRE) urine specimens harbor molecular signatures of aggressive PCa, which can help in the clinical management of the disease. Previously, we had systematically evaluated urinary glycoproteins associated with aggressive prostate cancer (AG-PCa) using post-DRE urine specimens. The goal of the study is to explore the potential of using pre-DRE urine specimens for detecting AG-CaP and validate the post-DRE findings. Methods: This is a retrospective case cohort study based on 154 pre-DRE urine specimens collected by the Center for Prostate Disease Research under an institutional review board approved protocol from men undergoing diagnostic biopsy. We performed an optimized quantitative mass spectrometric analysis to evaluate the association of markers with aggressive PCa at diagnosis. The cohort comprised 41 patients with no cancer at biopsy, 48 patients with non-AG-PCa (Gleason score = 6), and 65 patients with AG-PCa (Gleason score 7 or above). Targeted analyses of AG-PCa-associated glycoproteins previously identified in post-DRE urine samples were also applied to validate the post-DRE findings in non-DRE specimens. Data-independent acquisition mass spectrometry (DIA-MS) analysis was performed for glycosite-containing peptides. Fisher exact and Wilcoxon-Mann-Whitney tests were used for categorical and continuous variables respectively and logistic regression was evaluated using receiver operating characteristic (ROC) analysis for the urinary markers and marker panels. Results: Urinary prostatic acid phosphatase (ACPP), CD97 antigen (CD97), and prostate-specific antigen (PSA) levels in pre-DRE specimens were associated with patients with AG-PCa compared to cancer-negative and non-AG-PCa. The analysis confirmed three of the previously reported post-DRE urinary glycoproteins and identified three new AG-PCa-associated glycoproteins including Fibrillin 1 (FBN1), Vitronectin (VTN) and Hemicentin 2 (HMCN2). A multi-marker panel composed of urinary PSA, VTN, HMCN2, CD97 achieved an AUC of 0.78 (p= 2.69 x 10-9) for distinguishing AG-PCa from cancer-negative & Gleason 6. Analysis of differentially abundant glycopeptides in pre- and post-DRE samples showed that compared to glycopeptides from the post-DRE urine data, humoral immunity-related proteins were enriched in pre-DRE urine samples, whereas immune cell-related proteins were enriched in post-DRE urine samples. Conclusions: Comparisons between post-DRE and pre-DRE urine specimens for glycoproteins have shown that pre-DRE samples hold promise for a non-invasive and simple early option for AG-PCa detection in patients. The identified AG-PCa-associated glycoproteins may be further evaluated in large cohort of pre-DRE urine specimens for detecting clinically significant prostate cancer. SOURCE OF Funding: NCI/EDRN IAA No. ACN 17005-001-0000011 (Gregory Chesnut)
.jpg "Gregory T. Chesnut, MD photo")