A MICRODISSECTION APPROACH TO DETECT MOLECULAR MARKERS DURING PROGRESSION OF PROSTATE-CANCER

To investigate the underlying mechanisms of carcinogenesis, we have de veloped a technique to determine the frequency of genetic changes in p rostatic carcinoma tissue. We have demonstrated that at a ratio of bet ween 1:4 and 1:9 mutant-normal alleles, the signal from a mutant TP53 allele is not apparent after polymerase chain reaction (PCR) amplifica tion and further direct sequencing or single-strand conformation polym orphism (SSCP) analysis. To bypass this problem, which is inherent in the heterogeneity of the prostate tissue and of the tumour, we selecte d areas of graded prostate tumours (Gleason score) from cryosectioned preparations, and microdissected these cells (20-100 cells). After ani onic resin removal of proteins, PCR amplification of TP53 gene exons 5 /6 and SSCP analysis, an abnormal SSCP band shift was observed in susp ected tumour cells, compared with microdissected stromal cells used as an internal control, while (1) a crude preparation of tissue DNA carr ying the tumour did not show any abnormality and (2) immunostaining by a set of monoclonal antibodies against TP53 protein remained negative . Nucleotide sequence analysis of the different bands confirmed the pr esence of a mutation in the TP53 gene exon 6 position 13 336 in an abn ormal band for one specimen, while no mutation was detected in the nor mal SSCP band. By targeting recognised tumour cells we can find DNA mu tations which are undetectable using the standard technique of whole-t issue DNA extraction, particularly in a heterogeneous tumour such as c arcinoma of the prostate.