Detection of p53 mutations in circulating DNA in serum/plasma in lung cancer patients

771 The p53 tumor suppressor gene is mutated in over 50% of all cancers. In addition to the high frequency of the mutation, the characteristic mutation spectrum in p53 is specific for particular cancers and exposures. Therefore, p53 mutation is a good target for investigating cancer. Recently, detection of genetic mutations, such as p53, K-ras, and N-ras, in serum/plasma DNA in cancer patients has been reported as a molecular biomarker. We are testing the hypothesis that specific p53 alterations in serum/plasma DNA are an effective biomarker of disease, exposure, or susceptibility in cancer patients and individuals who are cancer prone. Most previous studies focused on cancer patients and were not quantitative. A highly sensitive assay is necessary due to the low frequency of alterations in serum/plasma DNA. A modified form of the Mutation Load Assay, a highly sensitive, quantitative assay was developed for the detection of p53 mutations in serum/plasma DNA at codon 157, due to the specificity of the 157 mutation for cigarette smoking induced lung cancer, and at codons 248 and 249 due to the frequency of mutations at these positions. The Mutation Load Assay was determined to be highly sensitive. It was demonstrated, using the Mutation Load Assay, that mutations at codon 157(GTC>TTC), 248(CGG>CTG, CAG), and 249(AGG>AGT) were detected in serum DNA from individuals whose primary tumors contained the identical mutations. For these individuals, the mutation frequencies were estimated to be 10-3 - 10-4. After validation of the assay, it was applied to a pilot study of 12 cancer cases, 14 hospital controls, and 16 population controls. DNA was isolated from 600ul of plasma from participants and the p53 copy numbers was determined using real time PCR. The concentration of DNA as determined by estimation of p53 copy number was similar in cases, hospital controls, and population controls. Using 5000 p53 copies per sample, the mutation load assay was performed. In the pilot study, few samples were positive for mutations at codons 248 and 249 and the mutation frequency was low. Ongoing studies are evaluating reproducibility of low levels of detection and the effect of increasing the amount of p53 copies used in the assay.