Detection of one cell in a population of more than one million cells (rare event analysis) is increasingly important in biology. Detection and characterization of minimal residual tumor (MRT) during and after high dose cancer therapy is of particular importance: 1) MRT cells are survivors of an intense therapeutic barrage and represent a model of in vivo resistance; 2) Is the detection of MRT synonymous with relapse? 3) MRT in the autograft may contribute to relapse; 4) Microscopic tumor may represent an early metastatic event relevant as a prognostic marker and a signal for therapeutic intervention in early stage cancer. Detection of MRT is technically challenging given the rarity of abnormal cells (at the limits of sensitivity and specificity). Characterization of MRT is even more difficult since so few cells are available for study. This project will develop methods to detect MRT using in situ markers tagged with multiple fluorochromes (to enhance specificity and detect patterns of coexpression) examined by in situ RT-PCR and confocal fluorescence microscopy fitted with a computerized automated laser scanner. Using these techniques these cells can be directly visualized and isolated. We will adopt our newly developed palindromic PCR-cDNA display method for the identification of mRNAs with unique or increased expression by breast or small cell lung carcinomas but not normal marrow cells. Several new genes of interest have already been identified (including TC1, TC3, and TC4) overexpressed 5 to 50-fold in breast carcinomas. This method generates coding region cDNA sequences using rTth DNA polymerase and palindromic primers. Proteins derived from cDNA fragments can be rapidly expressed in bacteria and specific antibodies raised. We hope to identify mRNAs more abundantly expressed in minimal residual tumor to: 1) perform in situ RT-PCR for normal vs. minimal residual tumor; 2) determine full- length cDNA sequences of novel genes; and 3) generate additional antibodies for minimal residual tumor detection. Some genes unraveled by our approach may complement ongoing studies of oncogenes and tumor suppressor genes and aid in our understanding of the nature of minimal residual tumor. Our ultimate aim is to understand why tumor cells survive high dose therapy, and then discover targets to eradicate this tumor cell subpopulation.
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