Validation of quantitative fluorescence imaging analysis (QFIA) of β-catenin in archived prostate tissues for cancer biomarker discovery.

B16 Diagnosis of prostate cancer (CaP) depends on biopsy of individuals with prostatic nodules or persistently elevated serum PSA. Unfortunately, a serum PSA of 4-10 ng/mL has a specificity of 25% and biopsy has a false negative rate of 20%, requiring large-scale rebiopsy. A potential solution to this problem is based on the recognition of altered gene expression in normal-appearing acini (NAA) of cancerous prostate glands. Our goal is to evaluate β-catenin expression in NAA, as a potential biomarker for detection of CaP. In this study, we validated QFIA for quantification of β-catenin protein in slide specimens of prostate tissues fixed in formaldehyde. Stability of the imaging system was assessed prior to each image capture session. Reproducibility of β-catenin quantification by QFIA was established with LNCaP cells and sections from a benign hyperplastic (BH) prostate gland. Quantification of β-catenin was validated by reverse-phase protein array analysis (RPPA) of methacarn-fixed specimens from two BH and eight cancerous glands screened to provide a wide range of protein expression. Concordance of QFIA measurements of β-catenin in methacarn- and formaldehyde-fixed tissues was determined with sections from two BH and eight cancerous glands. Slide-mounted tissues and cells and protein arrays were labeled with epitope-saturating concentrations of primary antibody, followed by secondary antibody conjugated with AlexFluor® 488 (tissue/cells) or IRDye® 800CW (protein array). Protein signal was determined as mean pixel intensity (MPI) per cell or acinus and as integrated fluorescence intensity (IFI) per protein array spot. The imaging system exhibited remarkable stability. MPIs of standard fluorescent beads at 1, 10, and 100% relative intensity varied 5% or less for the 2000-hour life of the Hg/Xe excitation lamp. The average MPI of triplicate slides per analysis of LNCaP cells or standard BH tissue differed by 10% or less from the average MPI of all slides from multiple analyses. QFIA and RPPA measurements of β-catenin in adjacent tissue sections exhibited a strong linear correlation (r = 0.97), as did QFIA measurements of β-catenin in methacarn- and formaldehyde-fixed tissues (r = 0.86). Quantitative Fluorescence Imaging Analysis is an effective method for precise, reproducible quantification of β-catenin in archived prostate specimens and can be applied to evaluating β-catenin expression as a biomarker for CaP detection.