title: Performance of a phosphoflow assay to determine phosphorylation of S6 ribosomal protein as a pharmacodynamic read out for mTOR inhibition.
abstract: OBJECTIVE
  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).
  
  
  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 anti-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 (0.00-27.4μg/L). This effect was also confirmed in vivo after a single dose of everolimus to healthy volunteers (n=3). However, spiking WBS with 500μg/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 (<24h). 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.
  
  
  CONCLUSIONS
  The phosphoflow assay of p-S6RP performed well analytically, but sample stability, specificity, and method comparison results question its fitness for clinical purposes.
authors.affiliation: Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Kriegsbergstrasse 62, D-70174 Stuttgart, Germany. Electronic address: e.ahmed@klinikum-stuttgart.de.
authors.forename: Emaad
authors.initials: E
authors.lastname: Abdel-Kahaar
authors.affiliation: Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Kriegsbergstrasse 62, D-70174 Stuttgart, Germany. Electronic address: m.kabakchiev@klinikum-stuttgart.de.
authors.forename: Mariana
authors.initials: M
authors.lastname: Kabakchiev
authors.affiliation: MVZ Waiblingen, Praxis für Nieren- und Hochdruckerkrankungen, Beinsteiner Strasse 8/3, D-71334 Waiblingen, Germany. Electronic address: Hartmann.Bertram@mvz-gmbh.de.
authors.forename: Bertram
authors.initials: B
authors.lastname: Hartmann
authors.affiliation: Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Kriegsbergstrasse 62, D-70174 Stuttgart, Germany. Electronic address: e.wieland@klinikum-stuttgart.de.
authors.forename: Eberhard
authors.initials: E
authors.lastname: Wieland
authors.affiliation: Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Kriegsbergstrasse 62, D-70174 Stuttgart, Germany. Electronic address: m.shipkova@klinikum-stuttgart.de.
authors.forename: Maria
authors.initials: M
authors.lastname: Shipkova
mesh_terms: D015703:Antigens, CD / Q000276:immunology; D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D000068338:Everolimus / Q000494:pharmacology*; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D010766:Phosphorylation; D038601:Ribosomal Protein S6 / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors*
chemical_list: D015703:Antigens, CD; D007166:Immunosuppressive Agents; D038601:Ribosomal Protein S6; D000068338:Everolimus; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases
keywords: Biomarker; Everolimus; Phosphoflow; S6RP; Sirolimus; mTOR