Technical approach for the study of the genetic evolution of breast cancer from paraffin-embedded tissue sections

Abstract

We have optimized a technique that allows the study of numerous chromosomal loci (n = 20-50) from single paraffin-embedded tissue sections by 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 subjecting DNA to several cycles of amplification with a degenerate (universal) primer and then with specific microsatellite primers. This amplified 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 (chromosome 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 breast 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 levels of p53 protein accumulation detected by immunohistochemistry were also positive for mobility shifts in the SSCP analysis. We observed that chromosome 17 allelic losses and imbalance occurred at as early a stage as ductal carcinoma in situ (DCIS). Although in some cases we observed allelic losses or imbalance affecting the 17p13 region, close to the p53 locus, several of the tumors showed dissociation between such loss or imbalance and p53 mutation. Lobular carcinomas were predominantly disomic for chromosome 17 in contrast with ductal tumors, which often showed polysomy for chromosome 17. This comprehensive approach correlating the tumor subtype, its allelotype, with specific chromosome copy number and specific gene mutations and expression in preinvasive or early invasive breast cancer lesions will potentially provide information of relevance for a better understanding of the multistep mechanisms of breast carcinogenesis.

Allelic imbalance at chromosome 17p13.3 (YNZ22) in breast cancer is independent of p53 mutation or p53 overexpression and is associated with poor prognosis at medium-term follow-up

“…Although YNZ22 allelic imbalance may occur in the same tumour as p53 mutation (Chen et al, 1991;Cornelis et al, 1994; seven out of ten patients in this study) it is clearly no longer tenable to suggest that YNZ22 allelic imbalance occurs only in the presence of p53 mutation (Singh et al, 1993) and the two may in fact be quite dissociated (Chen et al, 1996).…”

An intronic variant of the TGFBR1 gene is associated with carcinomas of the kidney and bladder

“…21 A single 8 -10 m thick paraffin section was stained and manually microdissected for each tumor sample. Using a companion H&E-stained slide as reference, tumor cells were microdissected with a fine-point surgical blade (#11) under an inverted microscope.…”

“…Microdissected samples were deparaffinized (3 washes with xylene for 30 min each) and rehydrated in decreasing concentrations of alcohol. 21 DNA was extracted with Instagene chelex matrix solution containing 60 g of proteinase K in a shaking incubator at 37°C overnight, according to the manufacturer's instructions (Bio-Rad, Hercules, CA). Samples were boiled for 10 min, vortexed and centrifuged at 7,000g for 5 min.…”

Genomic Instability at Premalignant and Early Stages of Breast Cancer Development

“…Previous allelotypic studies have not implicated the short arm of D9S157 chromosome 9 as a frequent target for allelic losses in breast cancer W (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). But most of these earlier studies were based on RFLP analysis | by means of Southern blotting with a very limited number of molecular markers for chromosome 9p (6,14).…”

“…Allelic losses at variable frequencies have been Microsatellite Length Polymorphism and CDKN2 Deletion Analysis. reported for numerous chromosome subregions, the most common PCR was performed using 60 jIM individual end-labeled primer sets, 175 AIM being lp34-36, 1q23-32, 3p2l-25, 6q, 7q31, 11p 1 5 , 13q14, 16q, corresponding cold primer, 100-300 ng of template, 2 mM MgC1 2 , 300 AIM 17p13, 17q, and 18q (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). LOH 3 has been classically viewed as the deoxynucleotide triphosphates, 1 X Taq buffer (Promega), 10% DMSO, and indirect evidence for the possible existence of a tumor suppressor 1.5 units of Taq polymerase (Promega).…”