Susan M. Lubert

Outer breast quadrants demonstrate increased levels of genomic instability.

AbstractBackground: Theory holds that the upper outer quadrant of the breast develops more malignancies because of increased tissue volume. This study evaluated genomic patterns of loss of heterozygosity (LOH) and allelic imbalance (AI) in non-neoplastic tissues from quadrants of diseased breasts following mastectomy to characterize relationships between genomic instability and the propensity for tumor development. Methods: Tissues from breast quadrants were collected from 21 patients with various stages of breast carcinoma. DNA was isolated from non-neoplastic tissues using standard methods and 26 chromosomal regions commonly deleted in breast cancer were examined to assess genomic instability. Results: Genomic instability was observed in breast quadrants from patients with ductal carcinomas in situ and advanced carcinomas. Levels of instability by quadrant were not predictive of primary tumor location (P = .363), but outer quadrants demonstrated significantly higher levels of genomic instability than did inner quadrants (P = .017). Marker D8S511 on chromosome 8p22– 21.3, one of the most frequently altered chromosomal regions in breast cancer, showed a significantly higher level of instability (P = .039) in outer compared with inner quadrants. Conclusions: Non-neoplastic breast tissues often harbor genetic changes that can be important to understanding the local breast environment within which cancer develops. Greater genomic instability in outer quadrants can partially explain the propensity for breast cancers to develop there, rather than simple volume-related concepts. Patterns of field cancerization in the breast appear to be complex and are not a simple function of distance from a developing tumor.

Genomic patterns of allelic imbalance in disease free tissue adjacent to primary breast carcinomas.

Mammary stroma plays an important role in facilitating the neoplastic transformation of epithelial cells, modulating integrity of the extracellular matrix, and maintaining genomic stability, but molecular mechanisms by which stroma affects epithelial structure and function are not well-defined. We used laser-assisted microdissection of paraffin-embedded breast tissues from 30 patients with breast disease and a panel of 52 microsatellite markers defining 26 chromosomal regions to characterize genomic patterns of allelic imbalance (AI) in disease-free tissue adjacent to sites of breast disease and to define genomic regions that may contain genes associated with early carcinogenic processes. The mean frequency of AI in histologically normal tissue adjacent to the primary carcinomas (15.4) was significantly higher than that in distant tissue from the same breast (3.7). The pattern of AI across all chromosomal regions differed between the adjacent tissue and primary tumor in every case. Unique AI events, observed only in tumor (15 of informative markers) or only in adjacent cells (10 of informative markers), were far more common than AI events shared between tumor and adjacent cells (~ 4). Levels of AI characteristic of advanced invasive carcinomas were already present in non-invasive ductal carcinomas in situ, and appreciable levels of AI were observed in adjacent non-neoplastic tissue at all pathological stages. Chromosome 11p15.1 showed significantly higher levels of AI in adjacent cells (p < 0.01), suggesting that this region may harbor genes involved in breast cancer development and progression. Our data indicate that genomic instability may be inherently greater in disease-free tissue close to developing tumors, which may have important implications for defining surgical margins and predicting recurrence.

Analysis of Matrix Metalloproteinase-1 Gene Polymorphisms and Expression in Benign and Malignant Breast Tumors

A guanine insertion polymorphism in matrix metalloproteinase-1 promoter (MMP-1 2G) is linked to early onset and aggressiveness in cancer. We determined the role of MMP-1 2G on MMP-1 expression and breast cancer severity in patients with breast diseases. We observed no significant difference in genotype distribution among different disease groups. However, MMP-1 expression was significantly higher in atypical ductal hyperplasia than in benign breast disease and in invasive breast cancer compared to in situ breast cancer. MMP-1 2G insertion polymorphism in the invasive group also correlated significantly with the expression of MMP-1 and breast cancer prognostic markers HER2 and P53.

STR Analysis of Human DNA Samples After Dry-State Ambient Temperature Storage in GenPlates

Dry-state room temperature storage of DNA is of interest because it promises to be a relatively more cost effective and environmentally friendly method for transport and storage of DNA. However, this approach must be validated prior to large scale adoption by the scientist that currently store blood samples frozen. In this study, we compared the yield of DNA isolated from GenPlates™ to that from frozen blood, and verified the quality of the DNA by evaluating the success of genotyping and allelic assignment using the PowerPlex 16 system. Using 24 blood specimens from 6 human subjects, we determined that DNA yield from GenPlates™ a 96 well microtitre plate based product for transport and storage of DNA in the dry-state and at ambient temperature was 4.9% more than the yield from frozen blood after 16 months storage. The STR profiles from GenPlates™ DNA displayed 100% concordance with the profile from DNA isolated from frozen blood. Our results demonstrate that yield and PowerPlex 16 STR profile of DNA after dry-state ambient temperature storage in GenPlates™ for 16 months are comparable to DNA from frozen blood and 100% allelic assignment was achieved using 1 ng of DNA isolated from GenPlates™.

Abstract P4-07-09: The Impact of Matrix Metalloproteinase-1 Promoter 1G/2G Polymorphism on Breast Diseases

Background: Matrix Metalloproteinase-1 (MMP-1) is a ubiquitously expressed interstitial collagenase. Overexpression of MMP-1 has a role in initiating mammary tumorigenesis by degrading stroma and by releasing growth factors. A single guanine insertion polymorphism in the MMP-1 promoter creates the binding site, 59-GGAA-39, for the Ets transcription factor, and increases transcription of MMP-1. The MMP-1 2G polymorphism is linked to early onset, increased risk or aggressiveness of several cancers. Its relationship with other potential markers in invasion and metastasis of breast cancer is unknown. Experimental Design: To study the impact of the 2G polymorphism on breast cancer we analyzed the genotypes of 109 patients (52 invasive breast cancer [IBC], 29 ductal carcinoma in situ [DCIS], 13 atypical ductal hyperplasia [ADH] and 15 benign breast disease). Immunohistochemical (IHC) data for MMP-1, HER2, ER/PR and P53 from these donors were also analyzed. IHC results for MMP-1 were scored as 0 (no expression) or increasing expression of+1, +2 or +3. Data were analyzed using Pearson9s chi-square test to identify statistical significance. Results: A significantly higher number of patients in the IBC group expressed high MMP-1 (+2 and +3; p Conclusions: 1) In the IBC group, the 2G insertion polymorphism contributes to MMP-1 over expression. 2) Increased expression of MMP-1 in ADH and higher 2G allele frequency are consistent with the hypothesis that increased MMP-1 2G polymorphism plays a role in initiation of ADH through up regulation of MMP-1 expression. 3) Earlier studies show prognostic role for the coexistence of increased expression of HER2 and P53 in breast cancer. Our observation of a significant increase in the 2G homozygotes in HER2 and P53 positive patients supports a prognostic role for this polymorphism and suggests its possible association with other breast cancer markers. Thus, the MMP-1 2G polymorphism may both contribute to breast disease onset and serve as a prognostic marker for breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-07-09.