Comparison between the comet assay and the oxygen microelectrode for measurement of tumor hypoxia

Hypoxic cells are present in some solid tumours and are known to limit radiocurability. To compare two measures of tumour hypoxia, 25 patients with locally advanced disease and accessible tumours or metastatic nodes were examined using an oxygen microelectrode and the alkaline comet assay.

For the comet assay, fine needle aspirate biopsies were taken immediately following a dose of 5-10 Gy. Single cells were examined for radiation-induced DNA strand breaks, and the percentage of radio-resistant hypoxic cells within the population was calculated from DNA damage histograms. For oxygen tension (pO2) measurements, multiple tracks were made using an Eppendorf oxygen microelectrode. The possibility that application of the first method might influence hypoxic fraction measurement by the second method was examined in a more controlled system by creating four tracks in murine SCC-VII tumours using an oxygen electrode, and measuring hypoxic fraction at subsequent times.

For 28 tumours from 25 patients, hypoxic fraction measured by comet assay correlated with the percentage of PO2 values < 5 mmHg (r2 = 0.46, P < 0.001). The mean comet hypoxic fraction was 0.36 for five tumours with a median PO2 < 10 mmHg. For the remaining 23 tumours with a median PO2 > 10 mmHg, the mean hypoxic fraction was 0.09. Advancement of an oxygen electrode through SCCVII tumours had no significant effect on hypoxic fraction measured 5 min to 24 h later using the alkaline comet assay.

Tumours defined as hypoxic based on a median pO2 < 10 mmHg appear to contain more than 20% radio-biologically hypoxic cells as estimated by the comet assay. In an animal tumour model, puncture of the tumour with an oxygen electrode did not influence hypoxic fraction measured using the comet assay, in agreement with the clinical data that the order in which the two methods were performed was not important.