Preclinical and clinical evaluation of ɣH2AX as potentially predictive biomarkers for radiosensitivity


Preclinical and clinical evaluation of ɣH2AX as potentially predictive biomarkers for radiosensitivity

Meneceur, S.; von Neubeck, C.; Gudziol, V.; Krause, M.; Baumann, M.

Abstract

Introduction:

Ionizing radiation leads to DNA damages - including double strand breaks (DSB) - which are detected by DNA repair proteins. Upon DSB formation, the histone variant H2AX is phosphorylated - giving rise to ɣH2AX foci. GammaH2AX foci represent a promising biomarker to predict radiosensitivity - and might contribute to the individualization of cancer treatment in the future. It has previously been shown that residual ɣH2AX foci (24 h post irradiation) negatively correlate with local tumor control in head and neck squamous cell carcinoma (HNSCC) xenografts after 4 Gy irradiation in vivo [1]. Additionally, it is well known that the tumor micromilieu plays a critical role in cellular response to radiation. In this study, we propose to analyze residual ɣH2AX foci in established HNSCC models in a dose and micromilieu dependent manner [2]. Besides, the ɣH2AX foci assay has been translated to ex vivo irradiated patient biopsies. It could be shown that radiosensitive tumor entities present a steeper dose response curve compared to radioresistant entities [2]. Here, we show first results of the assay applied on HNSCC patient-derived biopsies.

Methods:

Cell line derived xenografts were subcutaneously transplanted on the hind leg of NMRI nude mice. When the tumor reached a size of 7x7 mm, BrdU (viability marker) and pimonidazole (hypoxia marker) were injected, and the mice were randomly distributed to 5 treatment arms (untreated control, 2 Gy, 4 Gy, 6 Gy, 8 Gy). The mice were sacrificed 24 h post irradiation, and the tumors were excised, fixed and embedded in paraffin (FFPE). Consecutive paraffin sections of the tumors were stained for BrdU and pimonidazole on the one hand - and for ɣH2AX on the other hand. GammaH2AX foci were manually counted in oxic areas at a maximal distance of 45 µm from the closest perfused vessel. In an attempt to transfer the assay to a clinical setting, patient-derived HNSCC biopsies were ex vivo cultivated for 24 h including 4 h of pimonidazole and BrdU treatment, subsequently irradiated with 0 – 8 Gy and FFPE fixed after 24 h.

Results and conclusion:

Preliminary results confirm that residual ɣH2AX foci increase in a dose dependent manner after in vivo irradiation, and that the different radiosensitivity of the xenograft models is associated with different slope of the dose response curves. In the patient materials, the different slopes of the dose response curves suggest patient specific repair capacities - which is of particular interest for treatment individualization.

Acknowledgement:

This work was supported by a grant of the Federal Ministry of Education and Research (BMBF 02NUK035C).

References:

[1] Koch et al., Radiother. Oncol. 108:434–39 (2013)
[2] Menegakis et al., Radiother. Oncol. 116:473–79 (2015)

  • Poster
    Gesellschaft für biologische Strahlenforschung, 26.-28.09.2016, Erlangen, Deutschland

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