Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho-protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma

Journal of Translational Medicine

Shidong Jia²  Priti Hegde²  Mark R Lackner²  Garret Hampton²  Allen Ebens²  Eric Humke²  Yibing Yan²  Marcin Kowanetz²  Matthew J Brauer²  Kathy Howell²  Doris Kim²  Jeff Eastham Anderson²  Dorothy French²  Joanna Lee²  Haider Mashhedi²  Basha Stankovich¹  Mahrukh Huseni²  Liangxuan Zhang²  Chikara Takahashi²  Congfen Li² 

[1] AllCells LLC, Emeryville, CA, 94608, USA;Genentech Inc, South San Francisco, CA, 94080, USA

关键词: Clinical trial;    Flow cytometry;    Meso Scale Discovery;    Immunohistochemistry;    Multiple myeloma;    Biomarker;    Pharmacodynamics;    Phosphatidylinositol 3-kinase;   

Others  :  827875
DOI  :  10.1186/1479-5876-11-76

 received in 2012-12-18, accepted in 2013-03-20,  发布年份 2013

【 摘 要 】

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.

【 授权许可】

   
2013 Li et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Hideshima T, Mitsiades C, Tonon G, Richardson PG, Anderson KC: Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets. Nat Rev Cancer 2007, 7(8):585-598.
• [2]Ikeda H, Hideshima T, Fulciniti M, Perrone G, Miura N, Yasui H, Okawa Y, Kiziltepe T, Santo L, Vallet S: PI3K/p110{delta} is a novel therapeutic target in multiple myeloma. Blood 2010, 116(9):1460-1468.
• [3]Steinbrunn T, Stuhmer T, Sayehli C, Chatterjee M, Einsele H, Bargou RC: Combined targeting of MEK/MAPK and PI3K/Akt signalling in multiple myeloma. Br J Haematol 2012, 159(4):430-440.
• [4]Younes H, Leleu X, Hatjiharissi E, Moreau AS, Hideshima T, Richardson P, Anderson KC, Ghobrial IM: Targeting the phosphatidylinositol 3-kinase pathway in multiple myeloma. Clin Cancer Res 2007, 13(13):3771-3775.
• [5]Baumann P, Schneider L, Mandl-Weber S, Oduncu F, Schmidmaier R: Simultaneous targeting of PI3K and mTOR with NVP-BGT226 is highly effective in multiple myeloma. Anticancer Drugs 2012, 23(1):131-138.
• [6]Munugalavadla V, Mariathasan S, Slaga D, Du C, Berry L, Del Rosario G, Yan Y, Boe M, Sun L, Friedman LS: The PI3K inhibitor GDC-0941 combines with existing clinical regimens for superior activity in multiple myeloma. Oncogene 2013. [Epub ahead of print]
• [7]Jia S, Roberts TM, Zhao JJ: Should individual PI3 kinase isoforms be targeted in cancer? Curr Opin Cell Biol 2009, 21(2):199-208.
• [8]Lackner MR: Prospects for personalized medicine with inhibitors targeting the RAS and PI3K pathways. Expert Rev Mol Diagn 2010, 10(1):75-87.
• [9]Jia S, Gao X, Lee SH, Maira SM, Wu X, Stack EC, Signoretti S, Loda M, Zhao JJ, Roberts TM: Opposing effects of androgen deprivation and targeted therapy on prostate cancer prevention. Cancer Discov 2013, 3(1):44-51.
• [10]Fuhler GM, Diks SH, Peppelenbosch MP, Kerr WG: Widespread deregulation of phosphorylation-based signaling pathways in multiple myeloma cells: opportunities for therapeutic intervention. Mol Med 2011, 17(7–8):790-798.
• [11]Liu Y, Hegde P, Zhang F, Hampton G, Jia S: Prostate cancer - a biomarker perspective. Front Endocrinol (Lausanne) 2012, 3:72.
• [12]Olmos D, Baird RD, Yap TA, Massard C, Pope L, Sandhu SK, Attard G, Dukes J, Papadatos-Pastos D, Grainger P: Baseline circulating tumor cell counts significantly enhance a prognostic score for patients participating in phase I oncology trials. Clin Cancer Res 2011, 17(15):5188-5196.
• [13]Brey EM, Lalani Z, Johnston C, Wong M, McIntire LV, Duke PJ, Patrick CW Jr: Automated selection of DAB-labeled tissue for immunohistochemical quantification. J Histochem Cytochem 2003, 51(5):575-584.
• [14]Krutzik PO, Irish JM, Nolan GP, Perez OD: Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications. Clin Immunol 2004, 110(3):206-221.
• [15]Darrah PA, Patel DT, De Luca PM, Lindsay RW, Davey DF, Flynn BJ, Hoff ST, Andersen P, Reed SG, Morris SL: Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major. Nat Med 2007, 13(7):843-850.
• [16]Krutzik PO, Trejo A, Schulz KR, Nolan GP: Phospho flow cytometry methods for the analysis of kinase signaling in cell lines and primary human blood samples. Methods Mol Biol 2011, 699:179-202.