1. Complete Panel of Monoclonal Antibodies Two further rounds of monoclonal antibodies (Mab) production will be completed under Phase II work. One round will use immunization by native rHuMGMT and selection by Western blot to denatured protein to select Mab with broader specificity. The other round will use immunization by formalin-fixed rHuMGMT to select Mab which may be useful in sectioned tumors by immunohistochemistry. 2. Development of a Qualitative Immunoassay Phase II work will focus on the development of the best method for immunodetection of MGMT in a variety of formats. The steps to be investigated include sample collection, processing, staining, and quantitation of Mab binding. Each Mab will be tested to determine the optimal reagent for cytological or histological tumor preparations. Improved sensitivity will be investigated using other fluorescent labels, such as Texas red and BIODPY. Quantitation of immunofluorescence will be accomplished by a computerized image analysis system. 3. MGMT Measurement in Brain Tumors Major brain tumor centers have been recruited to collect cytological or frozen sections, prospectively, and paraffin sections from patients, prospectively or retrospectively, and send them to Applied Genetics for MGMT quantitation by Mab. The tumor types selected for study at each center will be glioblastoma, astrocytoma or oligodendroglioma. The MGMT values together with patient and sample collection data will be collected for each treatment group. The statistical analysis will test the hypotheses that MGMT levels are inversely correlated either with time to relapse or time to death. Other corrections, such as response v. prior patient and/or treatment history, will also be examined.
