Login to Access Video or Poster Abstract: MP99-01
Sources of Funding: This study was supported by grants from the Princess Takamatsu Cancer Research Fund, JSPS KAKENHI Grant Number JP15K10588, and Osaka University project MEET.

Introduction

Extracellular vesicles (EVs) are microvesicles secreted from various cell types. EVs contain microRNAs, proteins, and mRNAs and play a role in intercellular communications via the mechanisms of exocytosis and endocytosis. We aimed to discover a new biomarker for high Gleason score (GS) prostate cancer (PCa) in urinary EVs via quantitative proteomics.

Methods

EVs were isolated from urine after massage from 18 men (negative biopsy [n = 6], GS 6 PCa [n = 6], or GS 8-9 PCa [n = 6]). EV proteins were labeled with iTRAQ and analyzed by LC-MS/MS. Candidate proteins were further analyzed by selected reaction monitoring/multiple reaction monitoring (SRM/MRM).

Results

Proteins extracted from EVs were enriched with CD9 protein, which is a marker of EVs, compared with unprocessed urinary proteins. EVs labeled with anti-CD9 antibody conjugated with Au colloids were also confirmed by electron microscopy. We identified and quantified 3530 proteins in the urinary EVs by LC-MS/MS. Thirty-six proteins increased in patients with PCa compared to those with negative biopsy (ratio > 2.0, p < 0.1). Four proteins increased in patients with GS 8-9 PCa compared to those with negative biopsy or GS 6 PCa (ratio > 2.0, p < 0.1). Twenty-seven proteins were chosen for further analysis and verified in 29 independent urine samples (negative [n = 11], PCa [n = 18]) using SRM/MRM. Among these candidate markers, fatty acid binding protein 5 (FABP5) was higher in the cancer group than in the negative group (p = 0.009) and was significantly associated with GS (p for trend = 0.011). Univariate logistic analysis showed that FABP5 was significantly associated with prostate cancers with GS 7 or more (p < 0.001). Even after adjusting for age, PSA, and PSA density, FABP5 was significantly associated with prostate cancer with GS 7 or more (p = 0.003). The receiver-operator characteristics curve analysis showed that the area under the curve (AUC) for the prediction of GS ? 7 by FABP5 was 0.856 (95% CI 0.708–1.00, p = 0.002), whereas the AUC value for prediction by serum PSA was 0.511 (95% CI 0.280–0.757, p = 0.87).

Conclusions

We applied the proteomic analysis to discover biomarkers in EVs in urine collected after prostate massage. FABP5 in urinary EVs could be a potential biomarker of high GS prostate cancer. Additional large-scale studies are warranted to confirm this finding.

Funding

This study was supported by grants from the Princess Takamatsu Cancer Research Fund, JSPS KAKENHI Grant Number JP15K10588, and Osaka University project MEET.