Evaluation of PUMA and NOXA expression as predictive biomarkers in prostate cancer.

Abstract

28 Background: PUMA and NOXA are two pro-apoptotic members of the BH3-only subgroup of the BCL-2 family. These two proteins play a role in the initiation of apoptosis. The objective of this study is to analyse their expression by immunofluorescence, alone or in combination, in benign and tumor prostate tissues to determine if there is a correlation between their expression and patient biochemical recurrence (BCR). Methods: Biomarker antibodies were verified for specificity and optimized by western blot and with tissue microarrays (TMA) containing prostate cancer cell lines and cell line derived xenograft tissues. Subsequently, quantification of expression for both biomarkers was performed on six TMA generated from radical prostatectomy samples (285 patients). The TMA were constructed using two cores of benign adjacent to the tumor and two cores of tumor tissue from each patient. Analysis of biomarker expression was semi-automated using the VisiomorphDP software. To optimize the analysis, we developed 2 different immunofluorescence masks: a cocktail of anti-cytokeratin-8 and -18 antibodies to identify epithelial cells and a combination of anti-p63 and anti-cytokeratin high marker weight to discriminate benign glands within tumor cores. Correlation with patient clinical outcome was determined with SPSS V20 software. Results: There was no correlation of PUMA and NOXA expression and BCR in tumor cores and stroma. In contrast, in benign epithelial cells Kaplan-Meier analysis showed a significant association between an extreme (low or high) PUMA expression and BCR (Log rank = 11.349, p = 0.001). Further analysis revealed a significant association between high NOXA expression in benign epithelial cells and BCR (Log rank = 6.133, p = 0.013). The combination of extreme PUMA and high NOXA identified patients with a poor prognosis (Log rank = 16.041, p = 0.000). In a multivariate Cox regression model, PUMA and NOXA proteins were also identified as independent predictive biomarkers of BCR. Conclusions: By studying benign epithelial cells adjacent to the tumor we identified two potential biomarkers that discriminate high-risk patients, independent of Gleason score or pathologic stage.