Next to surgery, radiation is the most effective treatment for malignant brain tumors. Nonetheless, -20% of glioblastoma multiforme (GBM) progress during the course of radiation therapy, suggesting that these lesions are resistant to radiation. Using tissues obtained during surgery, we will develop laboratory methodologies for identifying these tumors, and will quantify their response to radiation. Radiation sensitivity of mammalian cells is determined in the laboratory by measuring cell killing. However, for primary tumor tissue this approach is problematic because of low plating efficiencies and the resultant selection for study of a very small fraction of the original tumor cell population. Attempts to develop predictive assays based on brain tumor cell survival as the end-point have failed. We believe that it is crucial to determine whether other quantitative measures can be used to predict tumor response to radiation therapy. We propose to assess intrinsic radiation response with methods more suitable for use in primary tumor samples: measurement of DNA double strand breaks by pulsed field gel electrophoresis; NA supercoiling changes by the nucleoid halo assay; and measurement of chromosomal breaks and exchanges using fluorescence in situ hybridization techniques. We will determine hypoxic fraction in selected tumors using the comet assay. We will determine which of these assays provides relevant information regarding the radiation response of human brain tumor cells and establish predictive assays. Because of the problems inherent in studying radiation sensitivity of explanted tumors outside of their natural milieu, we will study the feasibility of irradiating malignant brain tumors in situ with a low energy X-ray source intraoperatively and harvesting tissue at intervals to measure DNA damage and repair and tumor hypoxia. This may be the ideal way to study the individual tumor's sensitivity to radiation.
Our specific aims are: 1) to define the optimal experimental conditions to quantify DNA damage and repair and hypoxic fraction in human brain tumor tissue grown as a xenograft model; 2) to determine which of the assays studied in Aim 1 show(s) the most promise as a predictive assay for in vivo radiation responsiveness in xenograft models; 3) to measure DNA damage and repair and chromosome breaks and exchanges in fresh GBM specimens using optimal conditions for the chosen assay(s); 4) to investigate the feasibility of irradiating human brain tumors at surgery with allow energy X-ray source and harvesting tumor tissue for assays of hypoxic fraction and DNA damage and repair; and, 5) to correlate the results obtained in Aim 3 with the tumor's BUdR labeling index and potential doubling time, genetic aberrations, activity of the DNA repair enzyme O/6-AT, and with established clinical indicators of prognosis, and clinical measures of radiation therapy outcome.
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