A comprehensive approach toward novel serum biomarkers for benign prostatic hyperplasia: the MPSA Consortium

Abstract

Purpose: Clinical benign prostatic hyperplasia is primarily diagnosed based on a diverse array of progressive lower urinary tract symptoms and is likely distinct from histological benign prostatic hyperplasia, which is detected by the presence of nonmalignant proliferation of prostate cells but may or may not be associated with symptoms. Pharmacological management of lower urinary tract symptoms has emerged as an effective initial treatment for clinical benign prostatic hyperplasia due to the introduction of new drug therapies shown to be effective in recent large clinical trials. Despite advances in symptom management and research into disease pathology, diagnostic strategies for the prediction of benign prostatic hyperplasia progression and response to drug modalities are lacking, and questions remain as to the molecular differences underlying clinical (symptomatic) vs histological (nonsymptomatic) benign prostatic hyperplasia.

Materials and methods: As part of the Medical Therapy of Prostatic Symptoms (MTOPS) clinical trial, which demonstrated the effectiveness of combination drug therapy in slowing benign prostatic hyperplasia progression, an archive of biological specimens linked to clinical data was collected for future profiling of disease pathology and changes associated with response to drug therapy. The MTOPS Prostatic Samples Analysis (MPSA) Consortium was established to identify and validate molecular markers that may better define benign prostatic hyperplasia related pathologies, identify risk of progression of lower urinary tract symptoms, and predict response to drug therapy using the MTOPS archive. The cooperating MPSA Biomarker Discovery Sites and Pathology Coordinating Center use diverse methodologies and scientific approaches as well as unique expertise to address the goals of the Consortium.

Results: To date the MPSA has identified a number of promising biomarkers as well as other molecular and cellular changes associated with benign prostatic hyperplasia.

Conclusions: These findings and ongoing Consortium discovery efforts have the potential to provide a greater understanding of the defects underlying disease pathology, and may lead to the development of early and more effective pharmacological treatment strategies for benign prostatic hyperplasia.

Figure 1. MPSA Strategy for BPH Biomarker Discovery and Validation

The MPSA Consortium’s three phase approach for development and/or validation for a given biomarker with associated benchmarks for progress and progression is depicted. Phase I represents the identification of promising candidates using local samples. Promising candidates are then moved to Phase II testing, which involves validation of candidates using larger local sample sets and common resources. Phase I markers may be moved directly into Phase III if they are exceptionally promising. Candidates validated in Phase II and that continue to show relevance for the group’s prioritized clinical questions are moved into Phase III testing, which involves analysis using banked MTOPS clinical samples. Putative biomarkers validated in Phase III would be candidates for future studies involving the development of tools for application in clinical and diagnostic settings.

Figure 2. The “Reverse Capture” Autoantibody Microarray Platform

Based on the ELISA dual-antibody sandwich immunoassay, an antibody microarray is used to immobilize native antigens corresponding to 500 pairs of unique, well-characterized monoclonal antibodies. Disease-related autoantibody reactivity to target antigens is then determined by scanning the microarray slides on which differentially labeled test and control IgG samples are competitively hybridized. The two-slide dye-swap method employed with every “reverse capture” autoantibody microarray experiment is also illustrated. The dye-swap allows for normalization of fluorescence and background intensity readings, as well as differences in labeling and antibody binding efficiency between the dyes. (Reproduced with permission from Proteomics Clin. Appl., epub ahead of print, April 19, 2007).

Figure 3. Antigen-Autoantibody Reactivities Using Different CyDyes

Each view represents the same autoantibody microarray in its entirety. (A) Antibodies from BPH patients with clinical progression were labeled with Cy3 dye (green), and patients without progression were labeled with Cy5 dye (red). Green spots highlighted by the ovals illustrate Cy3 > Cy5 and thus a distinct autoantibody profile for progression versus non-progression. (B) The CyDyes were reversed. Antibodies from BPH patients with clinical progression were now labeled with Cy5 dye (red), and patients without progression were now labeled with Cy3 dye (green). Red spots highlighted by the ovals illustrates Cy5 > Cy3 and, thus, illustrates the consistency of progression versus non-progression antigen-autoantibody reactivities when using different CyDyes.

Figure 4. Expression Array Analysis on Frozen Prostate Needle Biopsy Samples

The work flow for this MPSA pilot project is presented in this schematic figure. All cases are logged into the system using the MTOPS research ID code (A). Histologic evaluation of the samples occurs to determine the precise composition of each sample (B and C; reduced from X100). Some cases had predominantly stromal tissue (B) and other cases had more epithelium (C) requiring laser capture microdissection (LCM). In the pilot study, 23 biopsy samples had high enough stromal concentration that they did not require LCM (D; reduced from X1). 7 biopsy samples underwent LCM (data pending). After pre-processing, all samples undergo RNA extraction, RNA amplification, and finally cDNA expression analysis (E; reduced from X40).

Figure 5. DNA Microarray Profiles Reflect Transcriptomic Differences Between BPH Tissues and Between BPH and Normal Prostate Tissue

Expression data matrix of 241 genes (mRNAs) found with high expression (compared to reference channel) in MTOP BPH microarray profiles (average fold change>1.3), alongside the corresponding expression patterns for the genes in LCM profile data of stroma and epithelia from BPH and normal samples. See text for additional details.

Figure 6. MPSA Tissue Composition Analysis

Prostate needle biopsy stained with Masson’s trichrome combined with immunhistochemistry for epithelial cytokeratin (A) and subsequently colorized (B) to demonstrate glandular epithelium (purple), glandular lumina (green), stromal smooth muscle (red), and stromal fibroconnective tissue (blue). Reduced from X2.5.