Methods for the analysis of histone H3 and H4 acetylation in blood

Rigby, Lin; Muscat, Andrea; Ashley, David M.; Algar, Elizabeth

doi:10.4161/epi.20983

Cited by 19 publications

(17 citation statements)

References 24 publications

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“…25, 26 A representative example is shown in Supplementary Figure 1. Dynamic changes in histone acetylation from serial blood samples during cycle one from 17 patients are shown in Figures 2a and b.…”

Section: Resultsmentioning

confidence: 99%

Dual epigenetic targeting with panobinostat and azacitidine in acute myeloid leukemia and high-risk myelodysplastic syndrome

Tan

Wei

Mithraprabhu

et al. 2014

Blood Cancer Journal

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Therapeutic options are limited for elderly patients with acute myeloid leukemia (AML). A phase Ib/II study was undertaken to evaluate the maximum-tolerated dose (MTD) and preliminary efficacy of the pan-histone deacetylase inhibitor panobinostat (LBH589) in combination with azacitidine in patients with AML or high-risk myelodysplastic syndrome (MDS) naïve to intensive chemotherapy. Thirty-nine patients (AML=29, MDS=10) received azacitidine 75 mg/m2 subcutaneously (days 1–5) and oral panobinostat (starting on day 5, thrice weekly for seven doses) in 28-day cycles until toxicity or disease progression. Dose-limiting toxicities during the phase Ib stage were observed in 0/4 patients receiving 10 mg panobinostat, in 1/7 patients (fatigue) receiving 20 mg, in 1/6 patients (fatigue) receiving 30 mg and in 4/5 patients (fatigue, syncope, hyponatremia and somnolence) receiving 40 mg. In phase II, an additional 17 patients received panobinostat at a MTD of 30 mg. The overall response rate (ORR=CR+CRi+PR) in patients with AML was 31% (9/29) and that in patients with MDS was 50% (5/10). After a median follow-up of 13 months, the median overall survival was 8 and 16 months in patients with AML and MDS, respectively. Increased histone H3 and H4 acetylation was a useful early biomarker of clinical response. Combining panobinostat with azacitidine was tolerable and clinically active in high-risk MDS/AML patients, warranting further exploration.

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Section: Resultsmentioning

confidence: 99%

Dual epigenetic targeting with panobinostat and azacitidine in acute myeloid leukemia and high-risk myelodysplastic syndrome

Tan

Wei

Mithraprabhu

et al. 2014

Blood Cancer Journal

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“…Earlier studies had shown the presence of acetylated histones in blood cells, but its levels were not correlated with tumour tissue [27]. Such studies on global histone modifications are of importance because of their prognostic utility and predictive markers for recurrence.…”

Section: Discussionmentioning

confidence: 99%

A novel method for isolation of histones from serum and its implications in therapeutics and prognosis of solid tumours

et al. 2017

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BackgroundDysregulation in post-translational modifications of histones and their modifiers are now well-recognized as a hallmark of cancer and can be used as biomarkers and potential therapeutic targets for disease progression and prognosis. In most solid tumours, a biopsy is challenging, costly, painful or potentially risky for the patient. Therefore, non-invasive methods like ‘liquid biopsy’ for analysis of histone modifications and their modifiers if possible will be helpful in the better clinical management of cancer patients.MethodsHere, we have developed a cost-effective and time-efficient protocol for isolation of circulating histones from serum of solid tumor, HCC, called Dual Acid Extraction (DAE) protocol and have confirmed by mass spectrometry. Also, we measured the activity of HDACs and HATs in serum samples.ResultsThe serum purified histones were profiled for changes in histone PTMs and have shown a comparable pattern of modifications like acetylation (H4K16Ac), methylation (H4K20Me3, H3K27Me3, H3K9Me3) and phosphorylation (γ-H2AX and H3S10P) to paired cancer tissues. Profiling for the histone PTM changes in various other organs of normal and tumor bearing animal suggests that the changes in the histone PTMs observed in the tumor serum is indeed due to changes in the tumor tissue only. Further, we demonstrate that the observed hypo-acetylation of histone H4 in tissue and serum samples of tumor bearing animals corroborated with the elevated HDAC activity in both samples compared to normal. Interestingly, human normal and tumor serum samples also showed elevated HDAC activity with no significant changes in HAT activity.ConclusionsOur study provides the first evidence in the context of histone PTMs and modifiers that liquid biopsy is a valuable predictive tool for monitoring disease progression. Importantly, with the advent of drugs that target specific enzymes involved in the epigenetic regulation of gene expression, liquid biopsy-based ‘real time’ monitoring will be useful for subgrouping of the patients for epi-drug treatment, predicting response to therapy, early relapse and prognosis.

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“…Unlike these previous approaches, imaging flow cytometry as utilised in our study can also provide valuable information on localisation of the antibody:antigen interaction in WBCs, allowing identification of nuclear expression of acH4. This function allows us to measure histone H4 acetylation by quantifying the number of nuclear acH4‐positive cells, as compared to previously published techniques used to measure acH4, based on observing the expression of acH4 intensity . Our results suggest that nuclear acH4 positivity is a markedly more sensitive approach to measuring relative changes in acH4 expression as compared to mean and median intensity, although absolute numbers were lower.…”

Section: Discussionmentioning

confidence: 68%

“…This is the first assay which describes the quantification of acH4‐positive cells using an imaging flow cytometry approach, although previous studies have described methods to detect histone acetylation levels by conventional flow cytometry . Unlike these previous approaches, imaging flow cytometry as utilised in our study can also provide valuable information on localisation of the antibody:antigen interaction in WBCs, allowing identification of nuclear expression of acH4.…”

Section: Discussionmentioning

confidence: 99%

A novel imaging flow cytometry method for the detection of histone H4 acetylation in myeloid cells

Uitrakul

Hutton

Veal

et al. 2019

Eur J Clin Investigation

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Background The histone deacetylase inhibitor (HDACI) valproic acid has been shown to inhibit the growth of multiple paediatric tumour types and is well tolerated in a childhood cancer setting. The current study was designed to develop a novel imaging flow cytometry method for the detection of histone H4 acetylation in white blood cells obtained from childhood cancer patients treated with valproic acid. Materials and methods HL‐60 cells and whole blood samples from healthy volunteers were incubated with valproic acid (0‐8 mM) for 0‐24 hours, with additional blood samples collected from ependymoma patients receiving valproic acid on the SIOP Ependymoma II clinical trial. An imaging flow cytometry method was developed using an ImageStream®χ flow cytometer, collecting 100 000 images per sample following excitation of PE tagged acH4 antibody and DAPI. Results The mean percentage of acH4‐positive cells increased to a greater extent than increases in mean and median fluorescence intensity following incubation with valproic acid. Comparable results were observed for in vitro and ex vivo experiments, and the assay was shown to be appropriate for clinical sample analysis. Myeloid cells exhibited a smaller proportion of acH4‐positive cells than the lymphoid population, but a greater fold increase above basal levels. Conclusions The percentage of acH4‐positive myeloid cells has the potential to be used as a robust pharmacodynamic biomarker for the measurement of acH4 for HDACIs. The developed assay is now being utilised in a clinical trial involving the treatment of childhood ependymoma patients with valproic acid.

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Methods for the analysis of histone H3 and H4 acetylation in blood

Cited by 19 publications

References 24 publications

Dual epigenetic targeting with panobinostat and azacitidine in acute myeloid leukemia and high-risk myelodysplastic syndrome

Dual epigenetic targeting with panobinostat and azacitidine in acute myeloid leukemia and high-risk myelodysplastic syndrome

A novel method for isolation of histones from serum and its implications in therapeutics and prognosis of solid tumours

A novel imaging flow cytometry method for the detection of histone H4 acetylation in myeloid cells

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