Engrailed-2 (EN2): A Tumor Specific Urinary Biomarker for the Early Diagnosis of Prostate Cancer

Abstract

Purpose: Prostate cancer (PC) is the second most common cause of cancer related death in men. A number of key limitations with prostate specific antigen (PSA), currently the standard detection test, has justified evaluation of new biomarkers. We have assessed the diagnostic potential of Engrailed-2 (EN2) protein, a homeodomain-containing transcription factor expressed in PC cell lines and secreted into the urine by PC in men.Experimental Design: EN2 expression in PC cell lines and prostate cancer tissue was determined by semi-quantative RT-PCR and immunohistochemistry. First pass urine [without prior digital rectal examination (DRE)] was collected from men presenting with urinary symptoms (referred to exclude/confirm the presence of prostate cancer) and from controls. EN2 protein was measured by ELISA in urine from men with PC (n ¼ 82) and controls (n ¼ 102).Results: EN2 was expressed and secreted by PC cell lines and PC tissue but not by normal prostate tissue or stroma. The presence of EN2 in urine was highly predictive of PC, with a sensitivity of 66% and a specificity of 88.2%, without requirement for DRE. There was no correlation with PSA levels. These results were confirmed independently by a second academic center.Conclusions: Urinary EN2 is a highly specific and sensitive candidate biomarker of prostate cancer. A larger multicenter study to further evaluate the diagnostic potential of EN2 is justified.

Correlation of Urinary Engrailed-2 Levels to Tumour Volume and Pathological Stage in Men Undergoing Radical Prostatectomy

“…EN2 levels across the cohort were not normally distributed. Of the total of 57 patients in the cohort, 85% were positive for EN2 in their urine having levels above 42.5 ng/ml described previously) [6]. In these men, the mean EN2 level in urine was 347.73 ng/ml (median 265, IQR 184-524).…”

“…no diagnostic value). The threshold value for significance of EN2 level in urine was set at 42.5 ng/ml based on the original study [6].…”

“…First pass urine samples (10 ml), without prostatic massage, were collected a minimum of 4 weeks after any biopsy procedure (sextant or template) and, in general, were between 1 day and 2 weeks before RP. We previously reported that EN2 protein is stable in urine at room temperature for at least 4 days [6], and all urine samples were received for processing within 30 hours post donation. Upon receipt in the laboratory, samples were divided into 1.5ml aliquots, centrifuged 10,000 g for 5 minutes and the supernatant removed and stored at -80˚C.…”

“…A modified version of the previously published ELISA method [6] was used for this study. EN2 was bound directly to the surface of a plastic assay plate (Nunc Maxisorb) by incubating urine in each well for 2 hours.…”

“…We reported the potential diagnostic utility of Engrailed-2 (EN2), a transcription factor involved in embryonic brain development that is re-expressed in PC [6]. The presence of EN2 in urine was predictive of PC, with a sensitivity of 66% and a specificity of 88.2% (AUC of 0.81)…”