Performance of a phosphoflow assay to determine phosphorylation of S6 ribosomal protein as a pharmacodynamic read out for mTOR inhibition

Objectives: The S6 ribosomal protein (S6RP) is phosphorylated by the mammalian target of rapamycin (mTOR). The objective of this study was to assess the analytical suitability of a commercial kit-based phosphoflow cytometry protocol using whole blood (WBS) to measure the level of phosphorylated S6RP (p-S6RP) in T-cell subsets to study the pharmacodynamic effects of mTOR inhibitors (mTORi). Design and methods: A kit was used for fixation and permeabilization of mitogen-stimulated cells, and p-S6RP was assessed separately in CD3+ CD4+ and CD3+ CD8+ cells by employing an ariti-phospho-Ser235/236 antibody. Specificity, linearity, within-run precision and stability were investigated in either WBS spiked with everolimus and non-mTORi immunosuppressants or in WBS from patients on immunosuppressive therapy (n = 56). In addition, healthy controls (n = 10) and patients without immunosuppression (n = 10) were included. A comparison (n = 15) with an established western blot method based on anti-phospho p70S6 kinase (Thr389) was made by splitting WBS. Results: Everolimus decreased p-S6RP in vitro concentration dependently (0.00-27.4 mu g/L). This effect was also confirmed in vivo after a single dose of everolimus to healthy volunteers (n = 3). However, spiking WBS with 500 mu g/L cyclosporine also decreased p-S6RP. The within-run coefficient of variation was <18% in transplant patients and <27% in healthy controls for both cell subsets. Sample stability for p-S6RP analysis was limited (<24 h). p-S6RP was significantly decreased in CD3+ CD8+ cells of patients treated with sirolimus (p = 0.02) but not with everolimus. No significant correlation between the phosphoflow- and western blot method was noted. Conclusion: The phosphoflow assay of p-S6RP performed well analytically, but sample stability, specificity, and method comparison results question its fitness for clinical purposes. (C) 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.