Our objective is to develop a rapid and sensitive assay for in situ detection of receptor and relator c-onc mRNA in breast cancer. In situ is the method of choice because modest (3-5 fold) changes in per cell content of several key growth factor receptors can lead to selection and eventual domination of lineages with clinical relevance: for detection of occult aggressive cancers in benign disease; for diagnosis; for prognosis in mode- negative disease; and for predicting response to chemotherapy. The relatively small change in per cell content can be obscured by substantial intra- and inter-tumor heterogeneity when """"""""averaging"""""""" assays are used. Only radioactive methods give the required sensitivity for these low abundance mRNAs, and these methods are inferior to biotin/avidin-based methods for time, safety and other reasons. Lack of a sensitive and rapid biotin-based method has hampered transition of receptor research to routine clinical practice. Our studies indicate that insensitivity of in situ is due primarily to physical loss of mRNA from tissue during fixation and prehybridization steps. We propose a method that solves this problem by transferring mRNA directly from tissue cells in frozen section to immobilizing cellulose membranes. Preliminary data suggest that the RNA transfer is quantitative and that the resolution of the membrane transferred RNA, using biotinylated oliogo DNA probes, is approximately one cell diameter. The assays takes 7 hr. including a 4 hr. hybridization time. In Phase I we will validate the method for quantitating estrogen receptor, EGF receptor and c-ras in breast cancer; and, for resolving single receptor+ cells. This method would have wide application to the detection of low abundance mRNAs generally as well as small diffusible peptides, both in the research and clinical laboratory setting.
