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    Research Grants Awarded

    Array-Based Gene Methylation Profiling For The Characterization Of Breast Tumors In African-American And White Women

    Grant Mechanism:
    Investigator Initiated Research

    Scientific Abstract:
    In the United States, mortality due to breast cancer is highest among premenopausal African American women, compared with postmenopausal African American women and with whites. Microarray-based gene expression analyses have defined distinct breast tumor subtypes, referred to as the basal, luminal A, luminal B, and HER2+/ER- subtypes. We have characterized the breast tumors of younger and older African American and white women in the Carolina Breast Cancer Study (CBCS), a population-based, case-control study of invasive breast cancer in North Carolina for tumor subtype using immunohistochemical surrogates. The tumors of African Americans were more likely to be of the basal subtype, steroid receptor negative, highly proliferative, and have poorer prognosis compared with those of white women even when differences in stage at diagnosis were considered. The basis for these differences is not fully understood. In this proposal, we will further characterize the basal and other breast tumor subtypes in the CBCS using a new Illumina methylation microarray. Methylation, an epigenetic modification to DNA that does not alter the sequence code, occurs mainly at CpG sites within gene promoters and is a common mechanism of tumor suppressor gene silencing in cancer. Methylation of cancer-related genes is important in breast cancer because such changes can occur early, and specific patterns of methylation correlate with clinical features of breast tumors, response to endocrine therapy, and outcome. We hypothesize that promoter methylation profiles will differ among subsets of breast cancer defined by race, age, intrinsic breast tumor subtype (e.g., luminal A, basal, etc.), clinical prognostic features, etiologic risk factors, and outcome. We also hypothesize that certain subsets of breast tumors, e.g., basal tumors or p53 mutation-positive tumors, will show high levels of CpG site methylation in individual cancer-related genes, such as ER, or overall, may exhibit a hypermethylator phenotype. Our study is predicated on the idea that endogenous and exogenous factors affect promoter methylation and gene expression in susceptible breast epithelial cells, which in turn affect phenotype, clinical behavior and prognosis of breast cancers. To address our hypotheses, we will undertake the following Specific Aims: (1) Perform methylation profiling to measure methylation status of 1,505 CpG sites in the promoter regions of 807 cancer-related genes in 640 formalin-fixed invasive breast tumors from African American (n=254) and white women (n=386) in the CBCS using the new microarray-based Illumina GoldenGate Methylation assay. Statistical analyses will be conducted to explore (2a) Whether differences exist between the breast tumors of African American and white women in CpG site methylation patterns; (2b) Among African American and white breast cancer patients, whether CpG site methylation is correlated with clinical or tumor characteristics at the time of diagnosis including breast tumor subtype, and more specifically, whether new types of breast cancer are revealed by methylation analysis among the major tumor subtypes; (2c) If there exists within invasive breast cancers a hypermethylator phenotype, and if so, whether this phenotype is associated with intrinsic tumor subtype, survival, or other prognostic factors; (2d) Whether CpG site methylation profiles of breast tumors predict survival; and (2e) Whether CpG site methylation profile is correlated with etiologic risk factors, such as age at menarche or parity, or exposures such as smoking. This project builds on a well-characterized, population-based study of invasive breast cancer in African American and white women in North Carolina that includes extensive information on clinical characteristics, epidemiologic risk factors, tumor markers and breast cancer survival. Moreover, we have assembled a strong team of researchers with the expertise to ensure the successful completion of this study. Incorporation of a novel array-based technique for measuring methylation at 1505 CpG sites will provide a comprehensive assessment of methylation within 807 cancer-related genes. This study has high clinical-translational potential, offers a unique opportunity to significantly advance our understanding of breast cancer, and should lead to a reduction in incidence and mortality within a decade. It will provide new information on differential gene methylation patterns and the possibility of a hypermethylator phenotype that could impact clinical behavior, tumor subtype classifications and breast cancer prognosis. We expect that promoter methylation profiling will help to reveal new clinically-relevant subgroups within the major tumor subtypes, potentially leading to new therapeutic targets or prognostic markers for breast cancer. Because promoter methylation can be detected in early, pre-malignant lesions, and methylation analysis can be performed on formalin-fixed specimens, methylation profiling also has potential application to cancer diagnostics. Importantly, this study will improve our understanding of factors that contribute to disparities in breast cancer outcomes between African American and white women. Given the aggressive growth and poor survival associated with the basal subtype, undertaking further molecular characterization of this and other tumor subtypes is crucial. This project will yield additional insight into the biology of breast cancer subtypes, the etiology and progression of breast cancer, and inform our on-going study of racial differences in breast cancer among women in North Carolina.

    Lay Abstract:
    In the United States, mortality due to breast cancer is highest among premenopausal African American women, compared with postmenopausal African American women and with whites. Previous studies measuring gene expression identified distinct breast tumor subtypes, referred to as the basal, luminal A, luminal B, and HER2+/ER- subtypes. We have identified these breast tumor subtypes in younger and older African American and White women in the Carolina Breast Cancer Study (CBCS), a population-based, case-control study of invasive breast cancer in North Carolina, and found that the tumors of African Americans were more likely to be of the basal subtype, have poor prognostic features, and poorer prognosis compared with those of white women. However, the basis for these differences is not fully understood. In this proposal, we will further characterize the basal and other breast tumor subtypes in the CBCS using the new Illumina methylation microarray. Methylation, a modification to DNA that does not alter the sequence, occurs mainly at ?CpG sites? within regulatory regions of genes. Methylation is a common mechanism used to inactivate tumor suppressor genes in cancer, and methylation of cancer-related genes has been found to be related to clinical features of breast tumors, response to endocrine therapy, and outcome. We hypothesize that promoter methylation profiles will differ among subsets of breast cancer patients distinguished by race, age, breast tumor subtype, clinical features, breast cancer risk factors, and outcome. We also hypothesize that certain subsets of breast tumors, e.g., basal tumors or tumors exhibiting mutations in p53, will show methylation in individual cancer-related genes, such as ER, or may exhibit a hypermethylator phenotype and have high levels of methylation in many genes or CpG sites. To address our hypotheses, we will pursue the following Specific Aims: (1) Perform methylation assays to measure methylation at 1,505 CpG sites in the promoter regions of 807 cancer-related genes in 640 formalin-fixed invasive breast tumors from African American (n=254) and white women (n=386) in the CBCS using the new microarray-based Illumina GoldenGate Methylation assay. Statistical analyses will be employed to determine whether (2a) Differences exist between the breast tumors of African American and white women in CpG site methylation patterns; (2b) CpG site methylation is correlated with clinical or tumor characteristics including breast tumor subtype among African American and white women, and whether new types of breast cancer are revealed by methylation analysis among the major tumor subtypes; (2c) Some breast cancers have high levels of methylation at many genes, and if so, whether this ?hypermethylator? trait is related to tumor subtype, survival, or other prognostic factors; (2d) CpG site methylation patterns in breast tumors predict survival; and (2e) CpG site methylation profile is correlated with breast cancer risk factors, such as age at menarche or parity, or exposures such as smoking. The proposed study will offer a unique opportunity to advance our understanding of breast cancer. It will provide new information on gene methylation patterns that could impact clinical behavior, tumor subtype and prognosis, including the possibility that some breast tumors possess a ?hypermethylator? trait. Promoter methylation profiling will help to reveal new clinically-relevant subgroups within the major tumor subtypes, potentially leading to new therapeutic targets or prognostic markers for breast cancer. Because promoter methylation can be detected in pre-malignant lesions, and methylation analysis can be performed on formalin-fixed specimens, methylation profiling also has potential application to cancer diagnostics. Importantly, this study will increase our understanding of factors that contribute to disparities in outcomes between African American and white breast cancer patients. Given the aggressive growth and poor survival associated with the basal subtype, undertaking further molecular studies of this and other tumor subtypes is crucial. This project will yield new insights into the biology of breast cancer subtypes, the etiology and progression of breast cancer, and racial differences in breast cancer among women in North Carolina.