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

Abstract

15575 Background: The objective of this study was to develop a sensitive tissue proteomic assay for quantifying proteins (e.g. β- catenin) in tissues and prostate core biopsies by QFIA. A second objective was to apply the validated methodology to quantify other biomarkers for prostate cancer risk assessment, and to study oxidative stress in BPH, prostate cancer, the cancer field, and in inflammatory prostatitis in animals and human prostatitis. Methods: Biopsies from controls and cancer patients were depariffinized, the antigen retrieved, and labeled with a robotic BioGenex Stainer with a primary mouse monoclonal antibody (1/100) to β-catenin. A secondary goat anti-mouse IgG antibody (2° Ab 1/100) coupled with Alexa Fluor 568 was used as the indicator system. The Leica Fluorescence Microscope was calibrated and standardized with fluorescence beads, LNCAP cells, and BPH tissue sections. QFIA of β-catenin was validated by RPPA methacarn fixed tissues. Adjacent 4-micron tissue sections were analyzed by QFIA for total β-catenin content by RPPA. Results: Reproducibility was 10% or less for the LNCAP cells and BPH tissue controls. Adjacent tissue sections assayed by QFIA and RPPA exhibited a strong linear correlation (r=.97) as did tissues fixed in methacarn vs.10% buffered-formalin and assayed by QFIA (r=0.84). For the core biopsy specimens the average MPI (AMPI) from 40 to 200 acini was quantified. The AMPI of cancerous acini (CA) compared to nomal acini (NA) was reduced in 37 of 42 cases p<.02. ROC plots revealed that β-catenin expression in NAA identified 42% (95%CI 25 - 52%) of cancer cases, with 88% (95% CI 80%-96%) specificity. The tissue QFIA method was used to quantify oxidative stress biomarkers (MnSOD, HNE, 8-OHdG) and other field disease biomarkers (Connexin42, and UDP-glucose dehydrogenase (UGDH) in BPH, prostate cancer, and in prostatitis. Conclusions: A tissue based QFIA method has been developed for the quantification of β-catenin in prostate core biopsy specimens, and the method has been validated by RPPA. Assay of β-catenin in prostate core biopsy specimens shows promise as a potential biomarker for a profile defining individuals at risk for prostate cancer and studying oxidative stress in relation to the pathogenesis of disease. No significant financial relationships to disclose.