TECHNICAL APPROACH FOR THE STUDY OF THE GENETIC EVOLUTION OF BREAST-CANCER FROM PARAFFIN-EMBEDDED TISSUE-SECTIONS

We have optimized a technique that allows the study of numerous chromo somal loci (n = 20-50) from single paraffin-embedded tissue sections b y microsatellite length polymorphism analysis. DNA samples from normal and breast cancerous tissue can be obtained from the same section by means of microdissection. This technique was further improved by subje cting DNA to several cycles of amplification with a degenerate (univer sal) primer and then with specific microsatellite primers. This amplif ied DNA was also used to screen for mutations in the p53 gene by means of PCR-SSCP, In addition adjacent tissue sections were used to assess specific chromosome copy number by interphase cytogenetic analyses (c hromosome in situ hybridization) and to analyze expression of specific genes such as p53 and ERBB2. As an example of the use of our approach we performed a detailed chromosome 17 allelotypic analysis in 22 brea st tumors (5 ductal carcinomas in situ, 13 invasive ductal carcinomas, and 4 invasive lobular carcinomas), We detected mutations in the p53 gene by PCR-SSCP in 36% of the samples. Samples with significant level s of p53 protein accumulation detected by immunohistochemistry were al so positive for mobility shifts in the SSCP analysis. We observed that chromosome 17 allelic losses and imbalance occurred at as early a sta ge as ductal carcinoma in situ (DCIS). Although in some cases we obser ved allelic losses or imbalance affecting the 17p13 region, close to t he p53 locus, several of the tumors showed dissociation between such l oss or imbalance and p53 mutation. Lobular carcinomas were predominant ly disomic for chromosome 17 in contrast with ductal tumors, which oft en showed polysomy for chromosome 17. This comprehensive approach corr elating the tumor subtype, its allelotype, with specific chromosome co py number and specific gene mutations and expression in preinvasive or early invasive breast cancer lesions will potentially provide informa tion of relevance for a better understanding of the multistep mechanis ms of breast carcinogenesis.