The rs10993994 risk allele for prostate cancer results in clinically relevant changes in microseminoprotein-beta expression in tissue and urine

Abstract

Background: Microseminoprotein-beta (MSMB) regulates apoptosis and using genome-wide association studies the rs10993994 single nucleotide polymorphism in the MSMB promoter has been linked to an increased risk of developing prostate cancer. The promoter location of the risk allele, and its ability to reduce promoter activity, suggested that the rs10993994 risk allele could result in lowered MSMB in benign tissue leading to increased prostate cancer risk.

Methodology/principal findings: MSMB expression in benign and malignant prostate tissue was examined using immunohistochemistry and compared with the rs10993994 genotype. Urinary MSMB concentrations were determined by ELISA and correlated with urinary PSA, the presence or absence of cancer, rs10993994 genotype and age of onset. MSMB levels in prostate tissue and urine were greatly reduced with tumourigenesis. Urinary MSMB was better than urinary PSA at differentiating men with prostate cancer at all Gleason grades. The high risk allele was associated with heterogeneity of MSMB staining and loss of MSMB in both tissue and urine in benign prostate.

Conclusions: These data show that some high risk alleles discovered using genome-wide association studies produce phenotypic effects with potential clinical utility. We provide the first link between a low penetrance polymorphism for prostate cancer and a potential test in human tissue and bodily fluids. There is potential to develop tissue and urinary MSMB for a biomarker of prostate cancer risk, diagnosis and disease monitoring.

Figure 1. MSMB immunohistochemistry is specific for benign prostate glands.

MSMB immunohistochemistry was performed on TMAs using a BondMax autostainer. For all sections nuclei are shown in blue and MSMB staining in brown. (A) MSMB was not present in urothelium (left panel), black arrow - urothelium, white arrow - prostate, seminal vesicle (central panel), black arrow - seminal vesicle, white arrow - prostate, bladder (right panel), black arrow - fat cells, white arrow - bladder muscularis propria. (B) MSMB staining in the prostate is specific for benign glands (white arrows) and not prostate tumour cells (Gleason 3) (black arrows). MSMB also failed to stain atrophic benign glands. Examples of the staining criteria (none/weak, moderate and strong) applied to the immunohistochemistry. Results of the staining were highly significant as calculated using a Kruskal-Wallis test. n represents the number of pathological events scored.

Figure 2. The rs10993994 SNP correlates with MSMB protein expression in benign glands by immunohistochemistry.

(A) An example of mega-block staining of large prostate sections showing homogenous (left panel) or heterogeneous (right panel) staining. MSMB immunohistochemistry was scored as before as strong, moderate, weak or lost for each patient and stratified according to genotype; T - high risk, C- low risk. p-values were calculated using a Kruskal-Wallis test. n represents the number of pathological events scored.

Figure 3. The rs10993994 SNP correlates with urinary MSMB protein expression.

Urinary MSMB and PSA concentrations were determined and normalised to creatinine. (A) Urinary MSMB was determined by ELISA for the normal/benign patient cohort and a smaller cohort with a prostate cancer diagnosis. The same cohort was also tested for urinary PSA and serum PSA values collated. ROC curves were generated; AUC  =  area under the curve, confidence intervals are given in brackets and p values are given at 95% confidence levels. The difference between urinary MSMB and PSA ROC curves p = 0.0021. The difference between the urinary MSMB and The tumour cohort was further stratified by Gleason sum score (B). Urinary MSMB and PSA from low Gleason samples (6 and 7) were compared to high Gleason (8 and 9) samples and ROC curves generated and displayed as before. Urinary MSMB was also stratified according to the rs10993994 risk allele (C). n =  the number of individuals examined in each group.