Development of a quantitative immunofluorescent method for analysis of nuclear and cytoplasmic p53 and p21 in circulating tumor cells (CTC) as biomarkers of response to p53-targeted therapy.

Abstract

e13575 Background: The tumor suppressor p53 is involved in many aspects of cell cycle control and p53 mutations are the most common genetic abnormality in solid tumors. Several diverse approaches targeting p53 signaling including activators of p53, cell cycle checkpoint inhibitors and MdM2 inhibitors are under development and will require determination of p53 status to demonstrate proof of concept and ultimately drive patient selection. Measurement of p53 status in tumor biopsies is often hindered by limited availability of tissues. Detection of p53 status in CTCs offers an alternative to biopsy. Methods: A multiplex immunofluorescent (IF) method for identification of CTCs was developed using cytokeratin, CD45 and DAPI. DO7, a p53 specific antibody, and total and phospho-p21 antibodies were optimized for multiplex detection. Bleomycin (50 mU/mL) was used to activate p53 in A549 p53 wt cells, HT29 and SkBr3 p53 mutant cells. Single cell-based IF analysis was performed pre and post treatment by quantitative Laser Scanning Cytometry (LSC). Results: Wild type A549 cells expressed lower baseline levels of nuclear p53 compared to mutant p53 cell lines HT29 and SKBr3. Total and phosphorylated p21 was also lower in A549 cells compared to HT29 and SKBr3 cells. Bleomycin treatment of wt A549 cells led to a transient increase in p53 expression by 30% while mutant HT29 and SKBr3 cells did not show any change in p53. Treatment of A549 cells with bleomycin led to a 100% increase in total p21and a 70% increase in phosphorylated p21. No change in p21 was observed in p53 mutant HT29 and SKBr3 cell lines. Conclusions: We developed a sensitive, quantitative method for detection of nuclear and cytoplasmic p53 and p21 protein in cell lines that functionally discriminates between p53 wt and p53 mutant status. The method is transferable to circulating tumor cells (CTCs) and may have utility in selecting patients based on functional p53 status and predicting responses to p53 pathway targeted agents.