IDENTIFICATION OF P53 MUTATIONS IN ARCHIVAL PROSTATE TUMORS - SENSITIVITY OF AN OPTIMIZED SINGLE-STRAND CONFORMATIONAL POLYMORPHISM (SSCP) ASSAY

To correlate molecular changes with clinical information in prostate t issue, it is necessary to have accurate methods for screening for muta tions in clinically available specimens. We have refined the polymeras e chain reaction-single-strand conformation polymorphism (PCR-SSCP) an alysis for detection of p53 mutations in routine pathology specimens. These improvements help overcome technical barriers that interfere wit h SSCP analysis of archival tissues when only small amounts of poorly preserved formalin-fixed DNA are available. Furthermore, prostate samp les are heterogeneous in containing tumor, normal tissue, and hyperpla stic tissue. To address the first issue, the method included an initia l selection of PCR products using exonuclease I, followed by a second- step selection using nested PCR. This step ensures adequate amplificat ion of the target sequence while minimizing artifactual products that could otherwise interfere with mutation analysis. For the second issue , in addition to morphologic selection of appropriate tissue areas, we improved the sensitivity of detection of mutations by using restricti on enzyme digestion of products prior to SSCP analysis. Detection of m utations in heterogeneous tissue was evaluated by determining the mini mal detectable mutant allele frequencies in exons 4, 5, 6, 7, 8-9, and 10 by using mixtures of known mutant and wild-type cell lines, which were found to be 17.6, 9.1, 12.5, 8.1, 14.0, and 14.3%, respectively. To determine the utility of this method when used on heterogeneous cli nical samples, we performed a study of 19 archival prostate specimens (14 primary prostate cancers, three benign prostatic hyperplasia and t wo metastases) and detected abnormally migrating products in six of th e prostate cancer specimens (four primaries and two metastases). In fi ve of these samples, there was sufficient DNA to perform sequencing, w hich disclosed single-base change mutations in all five samples.