title: Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho-protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma.
abstract: BACKGROUND
  The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients.
  
  
  METHODS
  We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application.
  
  
  RESULTS
  The phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells.
  
  
  CONCLUSIONS
  We developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials.
authors.affiliation: Genentech Inc, South San Francisco, CA 94080, USA.
authors.forename: Congfen
authors.initials: C
authors.lastname: Li
authors.forename: Chikara
authors.initials: C
authors.lastname: Takahashi
authors.forename: Liangxuan
authors.initials: L
authors.lastname: Zhang
authors.forename: Mahrukh
authors.initials: M
authors.lastname: Huseni
authors.forename: Basha
authors.initials: B
authors.lastname: Stankovich
authors.forename: Haider
authors.initials: H
authors.lastname: Mashhedi
authors.forename: Joanna
authors.initials: J
authors.lastname: Lee
authors.forename: Dorothy
authors.initials: D
authors.lastname: French
authors.forename: Jeff Eastham
authors.initials: JE
authors.lastname: Anderson
authors.forename: Doris
authors.initials: D
authors.lastname: Kim
authors.forename: Kathy
authors.initials: K
authors.lastname: Howell
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Brauer
authors.forename: Marcin
authors.initials: M
authors.lastname: Kowanetz
authors.forename: Yibing
authors.initials: Y
authors.lastname: Yan
authors.forename: Eric
authors.initials: E
authors.lastname: Humke
authors.forename: Allen
authors.initials: A
authors.lastname: Ebens
authors.forename: Garret
authors.initials: G
authors.lastname: Hampton
authors.forename: Mark R
authors.initials: MR
authors.lastname: Lackner
authors.forename: Priti
authors.initials: P
authors.lastname: Hegde
authors.forename: Shidong
authors.initials: S
authors.lastname: Jia
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D014408:Biomarkers, Tumor / Q000378:metabolism; D001853:Bone Marrow / Q000378:metabolism; D001854:Bone Marrow Cells / Q000187:drug effects; D049109:Cell Proliferation; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005434:Flow Cytometry / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D007191:Indazoles / Q000494:pharmacology; D009101:Multiple Myeloma / Q000188:drug therapy*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: C532162:2-(1H-indazol-4-yl)-6-(4-methanesulfonylpiperazin-1-ylmethyl)-4-morpholin-4-ylthieno(3,2-d)pyrimidine; D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D004791:Enzyme Inhibitors; D007191:Indazoles; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D013449:Sulfonamides