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Metabolic Genotypes & Tumor Suppressor Gene Damage in Breast Cancer
Metabolic Genotypes and Tumor Suppressor Gene Damage in Breast Cancer.
Background: Breast cancer is the second leading cause of cancer related deaths among women in the United States. The relationship between metabolism of environmental carcinogens and endogenous estrogens and breast cancer is still under much scrutiny. Standard case/control studies have suggested that polymorphisms in drug metabolic enzymes that result in increased activation or decreased detoxification of carcinogens may be potential risk factors for breast cancer. However, the conflicting results and low levels of association that have been reported are probably due to the failure to identify the subset of patients that have mutations at key oncogenic loci. This suggests that breast cancer itself as an endpoint may not be the most appropriate biomarker for assessing the effects of polymorphic variants on risk for the disease. We thus propose to utilize a prospective case/case study design to determine the association between genetic damage to the p53 locus with the genotype of affected cancer patients for metabolic enzymes that play key roles in the metabolism of endogenous and exogenous human carcinogens.
Objective/Hypothesis: Breast cancer patients with polymorphisms in metabolic enzymes that may result in increased activation and/or decreased detoxification of exogenous and endogenous carcinogens will be more likely to have damage at tumor suppressor genes.
Specific Aims: (1) As part of a larger study, we will use blood samples from the first 500 patients to identify the allelic composition of the patient samples at the GSTM1, GSTT1, and GSTP1 loci. (2) Molecular screening of matched tumor samples will be utilized to identify the subset of patients harboring genetic damage at the p53 gene locus. (3) We will then determine whether an association exists between metabolic genotypes and genetic damage at the p53 locus in breast cancer patients.
Study Design: 500 breast cancer patients’ archived normal blood and tumor tissue along with medical history and dietary information obtained from questionnaires will be utilized in a case/case study design. DNA isolated from each blood sample will be used to determine the genotype of each patient following amplification by PCR and analysis by either agarose gel electrophoresis, restriction fragment length polymorphism (RFLP), or single stranded conformation polymorphism (SSCP). Laser capture microdissection will be used to isolate cancer cells from slides generated from the breast tumor samples. Isolated tissue will be used to screen the tumor samples for mutations in exons 4-9 of the p53 gene by PCR-SSCP analysis. Those samples with mutations will be sequenced to confirm the presence of the mutation as well as the location. Multivariable logistic regression will be used to determine whether an association exists between metabolic genotypes and genetic damage at the p53 locus in breast cancer patients. This coincides with a larger study in which cytochrome P450 genotypes will also be determined within a total population of 1,000 patients.
Outcomes and Benefits: The etiology of breast cancer is still not completely understood. Although both environmental and genetic factors have been implicated in breast carcinogenesis, attempts to determine an association between polymorphic variants in drug metabolic enzymes and risk for breast cancer have yielded conflicting results. We believe that the contradictory results reflect the fact that a suitable biomarker, such as mutated p53, must be analyzed as a marker of genetic damage that is potentially attributable and therefore likely to be associated with metabolic genotype. These data should provide critical information on the etiology and individual patient susceptibility to breast cancer induction, and may help identify patients at increased risk for breast cancer as a result of their complement of metabolic enzymes. This information could be further used in the development of chemopreventive agents or lifestyle changes.
Metabolic Genotypes and Tumor Suppressor Gene Damage in Breast Cancer.
The American Cancer Society estimates that approximately one out of every eight women are expected to develop breast cancer during their lifetime. In 2003, they expect 211,300 new diagnoses and 39,800 deaths as a result of breast cancer, making it the second leading cause of cancer related deaths among women in the United States. Prevention strategies are very limited, as the cause of breast cancer is still not completely understood. There are known risk factors for breast cancer that include the breast cancer susceptibility genes (BRCA1/2), family history, age, reproductive history, and exogenous factors that contribute to alterations in hormone levels. These risk factors account for less than half of the breast cancer cases diagnosed, stressing the importance of continuing well designed studies to determine the cause of the disease. Several studies support the role of environmental carcinogens and/or endogenous estrogens in breast cancer etiology. It appears that individual responses to environmental exposures can vary from person to person. This variation is based on polymorphisms in carcinogen and estrogen metabolizing genes such as glutathione S-transferases, or GSTs. These polymorphisms are genetic, inherited differences in DNA that result in alterations in the ability of individuals to metabolize carcinogenic and endogenous chemicals, resulting either in an increased ability to convert the compounds to a more toxic metabolite or a decreased ability to inactivate and eliminate the chemical. Carcinogens and/or endogenous estrogens may initiate cancer by causing mutations in critical genes that regulate cell growth, such as p53. Genetic differences in the ability of individuals to metabolize carcinogenic chemicals could thus influence the types of damage that occur in DNA. Epidemiological case/control studies hypothesizing an association between these polymorphisms and breast cancer risk have been very inconclusive, as there are studies that both support and refute this association. This may be caused by the large amount of genetic variation of individual breast cancer patients in the study populations. Our study intends to clarify the issue using a case/case, rather than a case/control, study design in order to identify the subset of patients that exhibit DNA damage indicative of exposure to carcinogenic insult. We hypothesize that breast cancer patients that have polymorphisms in metabolic enzymes that result in increased activation or decreased detoxification of environmental carcinogenic chemicals and/or endogenous estrogens will be more likely to have mutations in genes that regulate normal cell growth. The aims of this study begin with utilizing the blood sample and tumor tissue collected from 500 breast cancer patients. The blood samples will be used to determine if the patient exhibits genetic variation in three GST genes, GSTM1, GSTT1, and GSTP1. The tumor tissue donated by each patient will be analyzed for mutations in the tumor suppressor gene, p53. Statistical analysis will determine if there are significant associations between each or all of the polymorphisms and genetic damage in the tumor. Similar analyses will be used to determine if each of the polymorphisms are associated with specific types of mutations. The information gathered from this research can be used to identify women that are at increased risk for breast cancer and could most benefit from preventative measures either in the form of chemopreventive agents or lifestyle changes.