Polymerase chain reaction (PCR) technology has been adapted to permit ultra-sensitive quantitative determinations of relative levels of specific mRNAs and qualitative detection of mutations by RNA single-strand conformational polymorphism analysis (RNA-SSCP). These methods can be used on small tumor biopsy specimens and thus make it possible to analyze biochemical indicators of drug response in patients prior to therapy. In this study, we will establish biochemical determinants of response for locally advanced breast cancer that is to be treated with 5-fluorouracil (FU) by continual infusion in the first stage of therapy followed by FU plus radiation. The gene expression of thymidylate synthase (TS) will be measured as a candidate response determinant for FU by quantitative PCR. Previously we found a wide range of variation of 50-fold or more in TS expression among tumor specimens, including breast tumors. We will study whether this variation stems from differences in cell cycle distribution or abnormal regulation of genes in tumor cells. To study heterogeneity of TS expression among tumor cells, TS expression in infiltrating normal tissue, and per-cell expression between high and low expressing specimens, we will use in situ PCR and immunohistochemical methods. The p53 status of the tumors will be evaluated as a candidate response determinant for the radiation treatment. Screening for p53 mutations will be done by RNA- SSCP. With advanced breast cancer, it will be possible to measure response determinants in multiple tumor specimens from the same patient, including the primary tumor, metastatic sites, and multicentric sites within the breast after mastectomy. Since various tumor sites within the same individual often respond differently, the response is more likely due to inter-tumoral rather than inter-individual differences. The broad objective of this project is to establish whether analysis of tumor specimens for quantitative measurement of biochemical determinants can be useful and practical for predicting tumor response on an individual basis.
