Multi-omics biomarker pipeline reveals elevated levels of protein-glutamine gamma-glutamyltransferase 4 in seminal plasma of prostate cancer patients

Purpose: Prostate-specific antigen, a blood serum biomarker of prostate cancer, lacks specificity and prognostic significance, so considerable efforts are devoted to developing novel biomarkers. Seminal plasma, due to its proximity to prostate, is a promising fluid for biomarker discovery and non-invasive diagnostics. In this study, we investigated if seminal plasma proteins could increase specificity of detecting primary prostate cancer and discriminate between high- and low-grade cancers. Experimental Design: To select 148 most promising biomarker candidates, we combined proteins identified through five independent data mining or experimental approaches: tissue transcriptomics, seminal plasma proteomics, cell secretomics, tissue specificity and androgen regulation. A rigorous biomarker development pipeline based on targeted proteomics assays was designed to evaluate the most promising candidates. Results: We qualified 77 and verified 19 proteins in seminal plasma of 67 negative biopsy and 155 prostate cancer patients. Verification revealed a prostate-specific, secreted and androgen-regulated protein-glutamine gamma-glutamyltransferase 4 (TGM4), which could predict prostate cancer on biopsy and outperformed age and serum PSA. Machine-learning approaches also revealed improved multi-marker combinations for diagnosis and prognosis. In the independent verification set measured by in-house ELISA, TGM4 was up-regulated 3.7-fold (P=0.006) and revealed AUC 0.66 for detecting prostate cancer on biopsy for patients with serum PSA≥4 ng/mL and age≥50. Low levels of TGM4 (median 120 pg/mL) were detected in blood serum, but could not differentiate between negative biopsy, prostate cancer or prostate inflammation. Conclusions: Performance of TGM4 warrants its further investigation within the distinct genomic subtypes and evaluation for the inclusion into emerging multi-biomarker panels.