MOLECULAR APPROACH TO RAPID ASSESSMENT OF P53 TUMOR-SUPPRESSOR MUTATIONS IN ESOPHAGEAL TUMORS FROM STAINED HISTOLOGICAL SLIDES

The analysis of the tumor suppressor gene, p53, is of fundamental impo rtance in prognosis and staging in many cancers; however, the molecula r techniques required to analyze this gene have been expensive, time c onsuming, and unrelatable to the histological appearance of the sample s. This research explored one model of clinically testing for specific mutations in the p53 gene by scraping selected areas of stained histo logical slides and analyzing for ''hot-spot'' p53 mutations. Selective ly removing samples from the stained histological slide will be of spe cial value in examining suspicious regions in adenomas, potential meta static regions, and the margins of resected area. A polymerase chain r eaction (PCR)-mediated restriction fragment length polymorphism (RFLP) analysis approach in which naturally occurring or primer-mediated mut agenesis-induced restriction enzyme sites were utilized to test seven hot-spot mutations. These assays were able to detect one mutated seque nce in 100, and therefore, were sufficiently sensitive to be used with very heterogeneous tumors. Several of the assays could be multiplexed to reduce the number of PCRs necessary to screen for the seven mutati onal hot spots. Furthermore, an exact determination of the base change could be obtained by direct sequencing of the PCR products. Although this form of analysis may be applicable only to certain types of cance rs (e.g., bladder, brain, colon, esophageal, gastric, thyroid, and ova rian tumors), this approach can obtain detailed mutational information from specific regions of a histological slide in a cost-effective and timely manner.