Article Abstract

Quantification of DNA double-strand breaks in peripheral blood mononuclear cells from healthy donors exposed to bendamustine by an automated γH2AX assay—an exploratory study

Authors: Stefan Rödiger, Marius Liefold, Madeleine Ruhe, Mark Reinwald, Eberhard Beck, P. Markus Deckert

Abstract

Background: DNA double strand breaks (DSBs) are the most severe form of DNA damage in eukaryotic cells treated with ionizing radiation or chemotherapeutic drugs. They can be quantitatively assessed by fluorescence imaging of phosphorylated histone protein H2AX (γH2AX), where the number of γH2AX foci represents the number of DNA DSB. Real-time assessment of DSB could help tailoring cytotoxic therapies to individual patients regarding both response and adverse events. This would require reliable automated quantification technology not yet routinely available. Here we explore this concept in the context of malignant lymphoma.
Methods: To investigate the DSB response to cytotoxic treatment in vitro, peripheral lymphocytes of healthy donors were incubated with bendamustine, an alkylating drug commonly used in lymphoma therapy. To mimic the clinical setting, the drug concentration per number of donor cells was either calculated as a standard dose or based on the body surface area of the individual donor. DNA DSB were quantified by an automatized immunofluorescence γH2AX assay using the AKLIDES NUK® system.
Results: Across all donors, the mean number of γH2AX foci per cell was 1.29, IQR (1.08) after bendamustine treatment as opposed to 0.04, IQR (0.125) in untreated freshly isolated peripheral blood mononuclear cells (PBMCs). The standardized incubation dosage resulted in a mean of 0.89, IQR (0.51) foci per cell, while individualized dose calculation yielded 1.57, IQR (0.5) foci per cell. The difference in γH2AX foci between the two dosage calculations was significant (P=0.036). In addition, we observed a trend towards a negative correlation between the donors’ body surface area and the number of foci per cell. Between donors, no significant correlation of the number of foci in response to a given dose was observed. Dose titrations on the cells of individual donors demonstrated a significant response (P<0.05) between dose of bendamustine and the number of γH2AX foci per cell.
Conclusions: The automatic AKLIDES analysis platform can assess γH2AX foci for routine use. The individual γH2AX foci response to in vitro bendamustine is dose-dependent and can be monitored timely. Calculating individual in vitro dosage from the donor’s body surface area resulted in a broad variation of foci counts between individuals, suggesting that this dosage method does not result in equivalent biological effects among different individuals. Adjusting the dose individually based on biological responses such as DNA DSB could offer a way of personalized medicine with conventional substances, reducing toxicity while increasing therapeutic efficacy.