Our specific aim is to develop highly sensitive molecular methods for detecting early stage tumors by screening urine and other body fluids for the presence of cancer cells. We are attempting to detect somatic DNA changes associated with malignancy. Methods were developed to amplify DNA segments by the polymerase chain reaction (PCR) and detect disease-related mutations by single-strand conformational polymorphism (SSCP) analysis. To make molecular analysis compatible with clinical laboratory testing, our method has implemented commercially available automated systems for rapid electrophoretic analysis and nonisotopic development of gels. Using PCR-SSCP analysis, we have screened for mutations in the human p53 tumor suppressor gene and the K-ras oncogene. To enhance the resolution of mutation detection, we demonstrated sequence-specific migration patterns of each of the K-ras mutations found in human cancers. The differential mobility properties of mutant DNA molecules have also allowed us to enhance the sensitivity with which particular mutations can be detected within mixed DNA populations. In an effort to improve the speed of DNA sequence analysis we are evaluating prototypic oligonucleotide arrays to probe for mutations in synthetic oligonucleotides and PcR products. With the advent of recent compelling evidence that telomerase activation is a specific manifestation of major epithelial cancers, we have established a telomerase assay. We have validated our telomerase assay with a number of tumor cell lines, and we are testing clinical samples so that telomerase testing may be implemented in patient care.
