The DSB Biomarkers Pilot Study


Session type:

Ketan Shah1,2, Ketan A. Shah2, Katherine Vallis1,2
1University of Oxford, Oxford, UK, 2Oxford University Hospitals NHS Trust, Oxford, UK


Biomarkers of the damage caused by radiation to tumours would help to guide adaptive radiotherapy treatment. DNA double strand breaks (DSB) are the most important lesion caused by irradiation, and are accompanied by the local phosphorylation of histone H2AX to form "γH2AX" at intranuclear foci. The magnitude and kinetics of γH2AX formation and repair have been correlated in vitro to tumour cell survival. Clinical assays of γH2AX in tumours are now being developed.


The objective of the DSB Biomarkers Pilot Study was to develop an immunocytochemical assay for γH2AX foci and automated quantification in clinical tumour fine needle aspirate (FNA) specimens. Patients with palpable tumours under treatment at the Oxford Cancer Centre were recruited, and consented to up to three FNAs on each of three occasions.


Eleven patients were recruited to the study, with tumour types including melanoma, squamous cell carcinoma and non Hodgkins lymphoma. We developed an assay to demonstrate γH2AX foci, along with automated quantification using the Gray Institute's TRI2 platform. The coefficient of variation among synchronous FNA specimens was 49%. Paired tumour specimens were obtained from three patients before and after the first fraction of a course of radiotherapy. Analysis of these specimens showed a significant increase in γH2AX foci, along with colocalisation with foci of 53BP1, another component of DSB signalling.


This is the first report of an immunocytochemical assay for γH2AX in tumour fine needle aspirate specimens. Such a method could be applied where repeated clinical tumour measurements are required, for example to follow the kinetics of γH2AX repair. The variation in the measurement may reflect assay and/or tumour cell heterogeneity, and would limit the sensitivity of a γH2AX foci FNA assay to detect small differences in DSB numbers.