P53 ABNORMALITIES IN PRIMARY PROSTATE-CANCER - SINGLE-STRAND CONFORMATION POLYMORPHISM ANALYSIS OF COMPLEMENTARY-DNA IN COMPARISON WITH GENOMIC DNA

Background: The reported frequency of mutation of the p53 tumor suppre ssor gene (also known as TP53) in human carcinomas of the prostate has varied widely, ranging from 3% to 42%, This variability may be a cons equence of tumor heterogeneity and/or the use of different methods of analysis, Since p53 mutation has been associated with clinical outcome for a number of cancer types, determination of its true frequency in primary carcinomas of the prostate is important, Purpose: The principa l aims of this study were as follows: 1) to validate the utility of de tecting p53 gene mutations by means of polymerase chain reaction-singl e-strand conformation polymorphism (PCR-SSCP) analysis of complementar y DNA (cDNA) (synthesized from prostate tissue RNA and 2) to study the concordance of RNA- and DNA-based PCR-SSCP assays in detecting p53 mu tations in individual tumor fragments, Methods: RNA and genomic DNA we re isolated by means of standard techniques from specimens of 19 carci nomas of the prostate, selected on the basis of p53 data obtained in a previous analysis of cDNA (indicating that 14 were mutant and five we re wild-type), RNA was converted into cDNA by means of reverse transcr iption (RT); the cDNA was then amplified by means of nonisotopic (i.e. , nonradioactive) PCR, and the PCR products were subjected to SSCP ana lysis in polyacrylamide gels (RT-PCR-SSCP analysis), Genomic DNA was e xamined by means of SSCP analysis of isotopically labeled ((PO4)-P-32) PCR products (DNA-PCR-SSCP analysis), In both approaches, the protein coding region of the p53 gene was divided into multiple, smaller frag ments for study, PCR products exhibiting abnormal migration in SSCP ge ls were subjected to direct nucleotide sequencing or to cloning and se quencing of multiple clones, Results: RT-PCR-SSCP and DNA-PCR-SSCP ide ntified p53 gene abnormalities in 15 of the 19 selected carcinomas, in cluding one previously reported to be wild-type for p53, Overall, PCR- SSCP analysis identified 18 p53 fragments with abnormalities; three ca rcinomas showed two abnormalities each, Six (33%) of the 18 abnormalit ies were detected by both RT-PCR-SSCP and DNA-PCR-SSCP, 10 (56%) were detected by RT-PCR-SSCP alone, and two (11%) were detected by DNA-PCR- SSCP alone, The 18 abnormalities were caused by 20 changes in the sequ ence of the p53 gene; in one carcinoma, double mutations in two indivi dual p53 exons were identified, Conclusions and Implications: PCR-SSCP analysis of both RNA and DNA allows the detection of more mutations t han the analysis of either alone, Some primary carcinomas of the prost ate contain more than one altered p53 gene, consistent with the possib ility of intratumoral heterogeneity of mutation of this gene, For comp rehensive analysis of p53 mutations in carcinomas of the prostate, and perhaps in other tumor tissues, SSCP analysis of cDNA should be used in combination with SSCP analysis of genomic DNA.