Elizabeth L. Schubert

Array Comparative Genomic Hybridization Analysis of Genomic Alterations in Breast Cancer Subtypes

In this study, we performed high-resolution array comparative genomic hybridization with an array of 4153 bacterial artificial chromosome clones to assess copy number changes in 44 archival breast cancers. The tumors were flow sorted to exclude non-tumor DNA and increase our ability to detect gene copy number changes. In these tumors, losses were more frequent than gains, and gains in 1q and loss in 16q were the most frequent alterations. We compared gene copy number changes in the tumors based on histologic subtype and estrogen receptor (ER) status, i.e., ER-negative infiltrating ductal carcinoma, ER-positive infiltrating ductal carcinoma, and ER-positive infiltrating lobular carcinoma. We observed a consistent association between loss in regions of 5q and ER-negative infiltrating ductal carcinoma, as well as more frequent loss in 4p16, 8p23, 8p21, 10q25, and 17p11.2 in ER-negative infiltrating ductal carcinoma compared with ER-positive infiltrating ductal carcinoma (adjusted P values ≤ 0.05). We also observed high-level amplifications in ER-negative infiltrating ductal carcinoma in regions of 8q24 and 17q12 encompassing the c-myc and c-erbB-2 genes and apparent homozygous deletions in 3p21, 5q33, 8p23, 8p21, 9q34, 16q24, and 19q13. ER-positive infiltrating ductal carcinoma showed a higher frequency of gain in 16p13 and loss in 16q21 than ER-negative infiltrating ductal carcinoma. Correlation analysis highlighted regions of change commonly seen together in ER-negative infiltrating ductal carcinoma. ER-positive infiltrating lobular carcinoma differed from ER-positive infiltrating ductal carcinoma in the frequency of gain in 1q and loss in 11q and showed high-level amplifications in 1q32, 8p23, 11q13, and 11q14. These results indicate that array comparative genomic hybridization can identify significant differences in the genomic alterations between subtypes of breast cancer.

Single nucleotide polymorphisms (SNPs) in the estrogen receptor gene and breast cancer susceptibility.

In order to evaluate the role of inherited variation in the estrogen receptor (ESR1) gene in human breast cancer, we determined intronic sequences flanking each ESRI exon; identified multiple SNPs and length polymorphisms in the ESR1 coding sequence, splice junctions and regulatory regions; and genotyped families at high risk of breast cancer and population-based breast cancer patients and controls. Of 10 polymorphic sites in ESR1, four are synonymous SNPs, two are nonsynonymous SNPs and four are length polymorphisms; five are novel. No ESR1 polymorphisms were associated with breast cancer, either in the high-risk families or the case-control study. We therefore conclude that inherited genetic variation is not a mechanism by which the estrogen receptor is commonly involved in breast cancer development.

A splicing mutation in RB1 in low penetrance retinoblastoma

Abstract The pediatric eye-tumor retinoblastoma is widely held as a paradigm of human cancer genetics and has been a model system for both the two-hit hypothesis of dominantly inherited cancer as well as for the concept of tumor-specific loss of constitutional heterozygosity to achieve expression of the tumorigenic phenotype. Familial retinoblastoma is usually inherited as an autosomal dominant disease with high penetrance and expressivity. In a small but significant number of families, however, retinoblastoma is inherited with greatly reduced penetrance and expressivity. In these families, retinoblastoma tumors occur relatively late, are often unilateral, and unaffected carriers may exist. We have identified a mutation in such a family that exhibited extremely low penetrance and expressivity. This mutation appeared to affect splicing of the mutant allele such that both a normal length RB1 mRNA and a truncated RB1 mRNA were expressed from the same allele.

Single Nucleotide Polymorphism Array Analysis of Flow-Sorted Epithelial Cells from Frozen Versus Fixed Tissues for Whole Genome Analysis of Allelic Loss in Breast Cancer

Analysis of allelic loss in archival tumor specimens is constrained by quality and quantity of tissue and by technical limitations on the number of chromosomal sites that can be efficiently evaluated in conventional analyses using polymorphic microsatellite markers. Newly developed array-based assays have the potential to yield genome-wide data from small amounts of tissue but have not been validated for use with routinely processed specimens. We used the Affymetrix HuSNP assay, composed of 1494 single nucleotide polymorphism sites, to compare allelic loss results obtained from both formalin-fixed and frozen breast tissue samples. Tumor cells were separated from normal epithelia and nonepithelial cells by dissection and bivariate cytokeratin/DNA flow sorting; normal breast cells from the same patient served as constitutive normal. Allele results from the HuSNP array averaged 96% reproducibility between duplicates and were concordant between the fixed and frozen normal samples. We also analyzed DNA from the same samples after whole-genome amplification (primer extension preamplification). Although overall signal intensities were lower, the genotype data from the primer extension preamplification material was concordant with genomic DNA data from the same samples. Results from genomic normal tissue DNA averaged informative single nucleotide polymorphism at 379 (25%) loci genome-wide. Although data points were clustered and some segments of chromosomes were not informative, our data indicated that the Affymetrix HuSNP assay could provide an efficient and valid genome-wide analysis of allelic imbalance in routinely processed and whole genome-amplified pathology specimens.

Inherited breast cancer: an emerging picture.

A role for BRCA1 and BRCA2 in the control of genome integrity easily fits a tumor suppressor model. It is well established that mutations in DNA repair genes lead to genomic instability (138). Genomic instability may directly lead to tumorigenesis by allowing for the accumulation of mutations in key cell cycle regulators (139). The studies summarized here suggest that BRCA1, BRCA2, RAD51. and BARD1 function as a biochemical complex. This complex apparently plays a role in one or more of the DNA damage response pathways. Experimental data suggest that BRCA1 and BRCA2 function as regulators of transcription. These observations highlight some of the fundamental questions that remain to be addressed in the study of the biology of these genes. Are the DNA repair and transcriptional regulatory functions of BRCA1 and BRCA2 related? BRCA1 and BRCA2 may maintain the integrity of the genome by regulating expression of genes directly involved in this process. Alternatively, if the functions are not related, which is required for suppression of tumorigenesis? Researchers also are grappling with another paradox. If BRCA1 and BRCA2 are ubiquitously expressed, why do mutations in BRCA1 and BRCA2 lead specifically to tumors primarily of the breast and ovary, as well as a limited number of other tissues to a lesser degree? Nothing to date has been revealed that would explain how alteration of the transcriptional regulatory function and or the DNA repair function ascribed to BRCA1 and BRCA2 would result in tumor specificity as both of these functions are essential to a broad spectrum of tissues. It is possible that BRCAI and BRCA2 may regulate genes expressed only in the breast and ovary. Similarly, there may be unidentified BRCA1 and BRCA2 co-factors that are active only in the breast and ovary and, therefore, are critical to tumorigenesis. All breast cancer is genetic, although only a small fraction of cases are attributable to inherited genetic predisposition. Most breast cancer is due to genetic alterations that are specific to breast epithelial cells, many of which remain unknown. Integration of genetic approaches into research designed to elucidate biological pathways of breast cancer tumorigenesis will ultimately lead to new information critical to the development of new tools for the diagnosis and treatment of disease.

The Retinoblastoma Gene and its Significance

The first human tumour suppressor gene, the Retinoblastoma Susceptibility gene (RB1) was first demonstrated in retinoblastoma, a rare paediatric eye tumour which has been studied extensively over the last century. Genetic studies of retinoblastoma have yielded unique insights into familial cancer syndromes and the mechanisms of oncogenesis by tumour suppressor genes such as the RB1 gene. In this view, we will summarize past research into the genetics of retinoblastoma that led to the discovery of the RB1 gene and discuss the influence these results have had on the field of cancer research. In addition, we will discuss current research into RB1 as it relates to cancer and its potential for new therapies.

A Previously Unknown Polymorphism Located within the RB1 Locus Only Present in Asian Individuals

Although the retinoblastoma susceptibility locus (RB1) spans some 180 kb in the human and has been fully sequenced, few polymorphisms within the locus have been identified and none have been shown to vary in allelic frequency in different populations. We have identified a previously unknown polymorphism within intron 18 of the retinoblastoma susceptibility gene that is present in Asians but not in the other ethnic groups examined. This polymorphism eliminates a Tsp5091 restriction enzyme site, making it easily detectable for use in linkage analysis and genetic population studies.