EXPLOITING TUMOR HYPOXIA--PREDICTING AND TESTING THE OUTCOME

The overall objectives of this project are two-fold. First, to investigate in human tumor xenografts the cellular and tissue characteristics that will determine the effectiveness of adding tirapazamine to fractionated radiation. Second, to evaluate methods to predict and to measure the extent of the potentiation of fractionated radiation therapy by tirapazamine. This project will serve as the laboratory underpinning of the clinical trial with tirapazamine and also the clinical study of the different methods of measuring hypoxia in human tumors. The two major hypotheses to be tested are first, that the potentiation of fractionated radiation by tirapazamine can be predicted prior to radiation therapy, and second, that the response of the tumor to this treatment can be measured soon after the beginning of the treatment. The prediction of the effectiveness of tirapazamine will be determined by measuring single- strand breaks in individual cells following a test dose of tirapazamine using the Comet Assay. The first hypothesis is based on our previous findings that cell killing of hypoxic cells underlies the potentiation of fractionated radiation by tirapazamine and that it is DNA damage caused by the metabolism of tirapazamine under hypoxic conditions that leads to cell killing. The second hypothesis, that the potentiation of radiation can be measured during therapy, is based on our previous work showing that stable chromosome translocations can be measured using FISH with whole chromosome probes and that these measurements may predict for the enhancement of radiation-induced cell killing by the addition of tirapazamine. An important question relevant to how tirapazamine should be given in clinical practice is whether tumor oxygenation improves when tumors shrink following treatment. If this is the case, it would suggest that tirapazamine should not be given towards the end of therapy, or after tumors have shrunk beyond a certain amount. This would allow higher and more effective doses to be concentrated in the first part of the treatment when the tumors were more hypoxic. We will investigate this question in a number of transplanted mouse and human tumors using various assays of hypoxic fraction. These measurements will allow us to directly relate various assays for tumor hypoxia with similar measurements made with the Eppendorf electrode and the Comet Assay in human tumors following shrinkage of the tumors during therapy. The tumors to be used in this investigation will be the HT29, HT1080, A431, FaDu, SQ-20B, and A543 human tumor xenografts in scid mice. This project will also interact in the identification of tumors with high and low hypoxia and in preparation of the human tumor xenografts A431, FaDu and SQ-20B, using the hypoxia marker EF-5 to label hypoxic cells in frozen tumor sections.