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Sources of Funding: Deane Prostate Health, Icahn School of Medicine at Mount Sinai. Both Shalini S Yadav and Kamlesh K Yadav are supported by the Prostate Cancer Foundation Young Investigator Awards.

Introduction

Given the present controversy surrounding PSA testing there have been renewed efforts to identify novel blood, urine, and genetic biomarkers for PCa. Using immunodetection-based methods, we previously observed elevated levels of pseudouridine (&[Psi]), an isomer of uridine, in AR-negative PCa cell lines and proposed &[Psi]’s use as a potential marker of aggressiveness. To this end, we analyzed several prostate cancer tissue micro arrays (TMAs) by immunohistochemistry (IHC) to assess the correlation between &[Psi] expression and disease diagnosis. We also designed an enzyme-linked immunosorbent assay (ELISA) capable of high-throughput quantification of &[Psi]RNA from blood, urine and tumor patient samples.

Methods

Paraffin-embedded TMAs (104 cores/92 cases total) with adjacent normal prostate tissue, including TNM, clinical stage and pathology grade, were purchased from US Biomax. An optimized protocol for IHC was performed and cell scoring and intensity staining for each core were visually assessed in collaboration with a Mount Sinai pathologist. Isolated total RNA was bound to Covalink plates (Thermo) according to manufacturer’s protocol and was incubated with anti- &[Psi] antibody. Biotinylated and streptavidin-HRP antibodies were used for signal amplification, while TMB was used for color development. Absorbance is proportional to levels of &[Psi] (in ng), and a standard curve generated from &[Psi]-oligonucleotides is used to calculate &[psi] in patient samples._x000D_ _x000D_

Results

Immunohistochemical analysis demonstrated a correlation between Gleason grade and proportional/intensity staining of &[Psi]. In representative cores, nearly 80% of tumor glands in Gleason 3+3 cores exhibited 2+ staining while &[Psi]-expression is 3+ in 100% of the tumor cells in Gleason 5+4 cores. Blood RNA isolated from patients with Gleason 7+ tumors contained on average 2.08 and 6.1-folds greater quantities of &[Psi] compared to Gleason 6 and 7 tumors, respectively (n = 2 per group).

Conclusions

Our results establish a clear relationship between &[Psi] expression and clinical advancement of disease. Further studies with larger patient cohorts as well as additional studies to elucidate the biological role of &[Psi] would give valuable insight into how its expression could influence the onset or progression prostate cancer.

Funding

Deane Prostate Health, Icahn School of Medicine at Mount Sinai. Both Shalini S Yadav and Kamlesh K Yadav are supported by the Prostate Cancer Foundation Young Investigator Awards.