Valproate has been used clinically as an anticonvulsant for the last 50 years and is one of the major drugs used in the management of epilepsy. More recently it has been observed that valproate also functions as a histone deacetylase (HDAC) inhibitor, and this has kindled interest in the drug as an anti-cancer agent. We have developed and validated an assay to measure the proportion of myeloid cells in peripheral blood with nuclei localised acetylated Histone H4 detectable by immunofluorescence and imaging flow cytometry. Whole blood samples from healthy volunteers were incubated for 4 hours with 0, 50, 150 or 300 μg/ml of sodium valproate at 37°C with agitation. Following ex vivo exposure the samples were transferred to Transfix stabilising tubes and stored at 4°C to mimic collection from the clinic. Leukocytes were isolated from the fixed and EDTA anti-coagulated blood by red blood cell lysis and centrifugation prior to permeabilisation with ice cold methanol and staining with PE conjugated anti-acH4 and DAPI. The stained cells were run through an Imagestream MkII imaging flow cytometer with excitation at 488 and 405nm. Images of myeloid and lymphoid cells were resolved by area of the cell and side scatter. An x/y plot of acH4 staining intensity over bright detail similarity between acH4 and DAPI staining was used to identify myeloid cells with nuclear acH4 staining.
Ex vivo incubation of whole blood samples with pharmacologically relevant concentrations of valproate resulted in a measurable increase in the proportion of myeloid cells with acH4 positive nuclei in 4/5 samples. The percentage of myeloid cells with acH4 positive nuclei increased from 0.01-0.29% in untreated samples to 0.75-7.0% following exposure to 300 μg/ml valproate. Validation experiments were carried out to assess variance due to staining and imaging of single incubations with 0 and 300 μg/ml valproate when processed independently. Coefficient of variation values were high, 110% (0.6 ± 0.67%) and 42% (3.1 ± 1.3%), respectively. However, the 5 fold increase in positive myeloid cells retained statistical significance (p < 0.05, t-test). We have developed a semi-quantitative cell based imaging method to identify increased histone H4 acetylation of myeloid cells following exposure to valproate. This assay will be used to detect valproate HDAC inhibition in myeloid cells as a surrogate tissue in children being treated as part of the European SIOP Ependymoma 2012 trial.
Citation Format: Jamieson David, Wai Wong, Gareth Veal. Development of a method to measure acetylated histone H4 in the nuclei of circulating myeloid cells as a surrogate tissue for the pharmacodynamics of HDAC inhibitors in the treatment of solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5374. doi:10.1158/1538-7445.AM2015-5374