Abstract 2583: Imaging flow cytometry assay development and validation for the detection of histone H4 acetylation in white blood cells
Authors: Uitrakul Suriyon , Veal Gareth James , Hutton Claire , Jamieson David 
Published: August 2018

Abstract
Introduction: Histone deacetylases have been identified as oncogenes in several cancer types, providing an attractive target for anticancer treatment. In this respect, the histone deacetylase inhibitor valproic acid has been shown to inhibit the growth of multiple paediatric tumor types and is well tolerated in children with refractory solid or CNS tumors.
Objective: The aim of the current study was to develop and validate a novel imaging flow cytometry method for the detection of histone H4 acetylation in lymphoid and myeloid cell populations, and to assess the applicability of the method to a clinical trial setting.
Method: HL-60 cells and whole blood samples from healthy volunteers were incubated with valproic acid (0.5-8 mM) for 0.5-24 hours, followed by RBC lysis for the whole blood samples and fixed with cold methanol. Additional blood samples were collected from patients with ependymoma who were receiving valproic acid as part of the SIOP Ependymoma II clinical trial. An imaging flow cytometry method was developed using an ImageStreamχ flow cytometer, collecting WBCs with excitation of PE conjugated acH4 antibody and DAPI. Data were collected using Inspire™ software and further analysed by Ideas™ software 6.2. Both in vitro and ex vivo experiments were repeated on at least 3 occasions.
Result: In the HL-60 cell line the mean percentage of acH4 positive cells was 1.98% in the vehicle control sample, increasing to 10.9-77.9% when treated with 0.5-8 mM valproic acid for 6 hours, with percentages of 8.7-49.0% observed following incubation with 4 mM valproic acid for 0.5-24 hours. Comparable data were generated in lymphoid and myeloid WBC populations following ex vivo incubation of whole blood samples with valproic acid. Increases in the percentage of acH4 positive cells were observed in samples collected at 4 hours post-administration in patients receiving valproic acid as compared to pre-treatment samples. Myeloid cells appeared to have a smaller proportion of acH4 positive cells than observed in the lymphoid population but a greater fold increase above basal levels.
Conclusion: A new assay for detection of histone H4 acetylation in WBCs by imaging flow cytometry has been developed and optimised. The assay showed increases in acH4 positivity in both in vitro and ex vivo experiments following exposure to valproic acid. The method can be used for the measurement of acH4 as a pharmacodynamic biomarker for histone deacetylase inhibitors in drug development and monitoring of drug efficacy in clinical trials.
Citation Format: Suriyon Uitrakul, Gareth James Veal, Claire Hutton, David Jamieson. Imaging flow cytometry assay development and validation for the detection of histone H4 acetylation in white blood cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2583.