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Organization of the Organ Systems Branch Current SPORE Programs Information for the Public PART Program Information for Applicants IntraSPORE Communications
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University of North Carolina at Chapel Hill
Overall Abstract The UNC SPORE in Breast Cancer's unique goals emphasize multidisciplinary translational research that spans and links the population, clinical, and basic sciences, and examines health disparities between African-American and Caucasian populations. The UNC Breast Cancer SPORE's primary objectives are to: Population Science
Clinical/Translational Science
Core Facilities
Development and Inter-SPORE Collaboration
Project 1 The Carolina Breast Cancer Study (CBCS) is a comprehensive, interdisciplinary investigation into the causes of breast cancer in African American and white women. A major focus of the CBCS is to understand genetic susceptibility. Our goal is to conduct the first comprehensive, population-based study of genetic risk factors for in-situ and invasive breast cancer in African American and white women. Our recent results have shown that germline mutations in BRCA genes are rare in breast cancer patients. Common inherited polymorphisms in metabolism genes play a role in a much larger number of cases. To date, we have investigated hormone metabolism, carcinogen metabolism, and cell cycle control. We propose here to expand our study of genetic susceptibility to include DNA repair genes, and potential interactions between these genes and exposure to environmental factors. Our preliminary data indicates that polymorphisms in the base excision repair gene, XRCC1, and the double-strand break repair gene, XRCC3, are susceptibility genes for breast cancer. During Phase 1 of the CBCS (1993-1996), 861 cases of invasive breast cancer and 790 population controls were enrolled. During Phase 2 (1996-2000), 890 cases of invasive breast cancer, 514 cases of in-situ breast cancer, and 1232 controls were enrolled. After enrolling additional controls, the total number of participants is expected to be 2265 cases and 2086 controls. African Americans comprise 44% of invasive cases and 21% of in-situ cases. Controls are frequency matched to cases based upon age and race. CBCS participants complete in-depth interviews covering known and potential risk factors for breast cancer. Blood samples are collected for extraction of lymphocyte DNA. DNA samples and exposure histories are available from 2147 cases and 2021 controls. The current proposal provides for statistical and laboratory analyses using previously collected DNA samples and exposure information from the CBCS. The Specific Aims are to conduct high throughput genotyping for single nucleotide polymorphisms in genes involved in the principal DNA repair pathways: Base Excision Repair (XRCC1, APE1, HOGG1, Polb); Nucleotide Excision Repair (XPA, XPD, XPF, ERCC1); Double Strand Break Repair (XRCC3, XRCC4); Non-homologous End Joining (DNA PK, DNA ligase I); Direct Repair (MGMT, AGT). Main effects for each genetic locus will be estimated, as well as the combined effects of multiple genes (gene - gene interaction) and the joint effects of genetic and environmental factors (gene - environment interaction). The study will provide important information regarding genetic susceptibility to breast cancer in African American and white women, and help to clarify the role of ionizing radiation, smoking and other environmental exposures in the etiology of breast cancer. In addition, we propose to extend our data collection and obtain updated treatment and outcome information on breast cancer cases. This information will allow us to evaluate novel hypotheses related to the role of DNA repair in response to adjuvant chemotherapy and radiation treatment, and to generate pilot data for subsequent grant applications.
Project 2 Estrogen is a major risk factor for breast cancer. The biological effects of estrogen are mediated primarily through the two estrogen receptors (ER), ER-alpha (a) and ER-beta (b). The hypothesis addressed in this proposal is that alterations in components of estrogen receptor signaling pathways occur during breast tumorigenesis, and that certain etiologic subsets of breast tumors will be defined by these alterations. The proposed study will evaluate several new markers related to estrogen signaling and responsiveness in the Carolina Breast Cancer Study (CBCS), a large population-based, case-control study of invasive and in situ breast cancer in African-American and white women. In a small pilot study, we have detected the ER-a (A to G at nucleotide 908) mutation recently reported by Fuqua et al (2000) at relatively high levels in invasive and in situ breast cancers. For the proposed study, we will carry out more comprehensive laboratory analyses to detect the ER-a A908G mutation in invasive and CIS breast tumors, assess its mRNA expression in an allele-specific manner, and localize the mutation to histologic sub-regions of tumors with both invasive and CIS components. We will evaluate the immunohistochemical expression of ER-b, ER-a, and the combined expression of ER-a and ER-b in invasive and CIS breast tumors. Finally, we will screen the germline of invasive cancer cases and controls for CYP19/aromatase genotypes. Using statistical analyses, we will: (1) Determine the prevalence of the ER-a A908G mutation, and expression of ER-b and/or ER-a in invasive and CIS tumors, and the relationship among these markers; (2) Examine correlations between the ER markers and breast tumor histologic or clinicopathologic characteristics as well as previously-identified somatic alterations, including p53 mutation or overexpression, HER-2/neu gene amplification or overexpression, and expression of Ki-67, E-cadherin and VEGF; (3) Examine the correlation of hormonal risk factors or CYP19/ aromatase or CYP17 genotypic variation with breast tumor subsets defined by each somatic ER marker; and (4) Assess the risk of ER-defined breast cancer subtypes associated with hormonal risk factors. As treatment and follow-up information becomes available for the CBCS cases, we also plan to assess the clinical significance of these ER markers with response to hormonal therapies and survival.
Project 3 Reactive oxygen species derive from a number of different sources, and traditional and putative breast cancer risk factors can be linked to the disease by an oxidative stress mechanism, for example: 1) steroid hormones, as well as some environmental organochlorines, are metabolized to reactive quinones and hydroquinones, which can directly damage DNA; 2) the metabolism of alcohol results in production of ROS and damage to DNA; 3) BRCA1 is needed for post-transcriptional repair of oxidative damage, indicating that oxidative stress may be an important risk factor for women with a family history of the disease; and 4) the inverse relationship noted with consumption of fruits and vegetables could be related to their being a source of antioxidant vitamins. Also, several markers of oxidative stress in a number of studies were higher in women with breast cancer and those at high risk than among non-diseased women. Endogenous factors affect not only the generation of ROS, but also an antioxidant response to them. We propose that interindividual variability in genes that encode enzymes with pro-oxidant and antioxidant activities will have an impact on the generation of ROS and ultimately on breast cancer risk. Furthermore, we hypothesize that risk associated with metabolic variability will be associated with exposure to exogenous factors that increase the likelihood of production of ROS. In this project, we propose to evaluate this hypothesis utilizing data from the Long Island Breast Cancer Study Project. DNA isolated from the blood samples donated by a large population-based sample of breast cancer case and control women will be assayed for three different types of ROS-related genetic polymorphisms including: regulatory regions of enzymes or processes that generate ROS (myeloperoxidase and tumor necrosis factora); those that prevent oxidative stress by neutralizing ROS (extracellular and manganese superoxide dismutase, glutathione peroxidase, catalase and glutathione S-transferase M1); and those that will affect ultimate levels of ROS generated by the metabolism of steroid hormones (catechol O-methyltransferase, glucuronosyltransferase). In addition to the large number of subject for whom samples are available for the laboratory analyses (n = 1087 cases and 1122 controls), comprehensive assessment of the subjects' environmental exposures have already been obtained in the parent study. Thus, we can also explore whether the genetic polymorphisms in 'at-risk' genotypes will affect associations between breast cancer risk and factors that are likely to be related through an oxidative stress mechanism, such as fruit and vegetable intake, reproductive and hormonal factors, alcohol consumption, and environmental contaminants (organochlorines and PAHs). Results from this project can be confirmed utilizing data from the Carolina Breast Cancer Study.
Project 4 Metastatic breast cancer is incurable with present therapy. Immunotherapy using autologous dendritic cells to stimulate tumor-specific CD8+ and CD4+ T cells offers a new avenue in the treatment of individuals with metastatic disease. In the past year, we have initiated a phase I/II trial designed to test the effectiveness of administering dendritic cells (DCs) pulsed with an altered peptide ligand from the HER-2/neu yprotein to women with metastatic breast cancer. Although only two patients have received at least three vaccinations, we have been encouraged that one of these patients has had a significant partial response to this treatment. In this project, we have brought together experts in the areas of structural immunology, dendritic and T cell immunology, and viral vaccines, to design a more comprehensive HER-2/neu-based vaccine. We will investigate in vitro and in vivo using animal models whether altering multiple epitopes from the HER-2/neu protein can induce a more significant T cell and anti-tumor effect compared to the single altered epitope in our current clinical trial. Additionally, we will perform preclinical work in vitro using human DCs and in vivo using transgenic mice, to test the effectiveness of a novel viral system, Venezuelan equine encephalitis replicon particles (VRP), with a natural tropism for DCs to target the entire HER-2/neu gene and cytokines to DCs. We will evaluate in transgenic mice whether the anti-tumor and T cell activity of DCs infected with VRP is greater than that found using peptide-pulsed DCs. In year four of this proposal, we will use the data generated from these studies to design a second-generation vaccine with broader anti-tumor and T cell activity to be used in the next clinical trial at the Lineberger Cancer Center.
Project 5: The two most effective classes of chemotherapeutic drugs in breast cancer are the anthracyclines and the taxanes. These drugs differ in mechanisms of action and mechanisms of resistance. Recent studies suggest that anthracycline sensitivity is strongly affected by tumor cell growth and proliferation, and specifically that the receptor tyrosine kinase HER-2 has an effect on response to anthracyclines. In contrast, taxanes appear to cause tumor cell death largely through apoptosis, and the ability of the cell to avoid apoptosis may mediate response to this class of drug. In preclinical and clinical studies, the HER-2 -targeting biologic agent trastuzumab synergistically augments taxane-induced cell killing, and this drug is now commonly added to taxanes in advanced breast cancer. In this prospective cohort of 120 breast cancer patients who will receive neoadjuvant anthracycline-based and taxane-based chemotherapy, tumor samples will be obtained specifically for research purposes at three time points: 1) prior to any chemotherapy, 2) following treatment with an anthracycline-containing regimen but before beginning a taxane-containing regimen (with or without trastuzumab), and 3) after all chemotherapy. These samples will be analyzed for several well characterized predictive markers for responsiveness to the two regimens (anthracycline-based, taxane-based) and will also be analyzed using cDNA microarray analysis to determine the molecular profiles of the tumors before and after each form of treatment. The aims of the study are: Aim 1: To collect serial samples from primary breast cancers prior to, during, and after conclusion of neoadjuvant chemotherapy with an anthracycline followed by a taxane with or without trastuzumab; Aim 2: To obtain pre- and post-chemotherapy axillary lymph node tissue to allow correlation of lymph node markers with primary tumor and with chemoresponsiveness; Aim 3: To evaluate predictive markers of anthracycline or taxane chemotherapy response, focusing on two biologic axes: genetic stability/proliferation and survival signals/apoptosis; Aim 4: To determine the gene expression profiles of the tumor samples collected in Aim 1 (see Project #6); Aim 5: To develop and validate promising new predictive markers that use DNA or RNA isolated from frozen specimens or formalin-fixed, paraffin-embedded samples, including the analysis of anti-apoptotic molecule NF-kB and related and regulated molecules (see Project #7); Aim 6. To expand this study in a proposed InterSPORE collaboration with four other institutions. This InterSPORE collaboration will be submitted separately as a supplemental grant. The design of this study is novel in the ability to measure responsiveness in primary breast cancer tissue and possibly in involved lymph nodes in the same patient during different regimens. The profiling of tumor markers and gene expression before and after specific noncross-resistant drugs will allow us to identify markers and patterns for targeting of specific forms of therapy to those patients likely to most benefit.
Project 6
Tumor classification is an essential part of the disease management process as it is used by the clinician to help guide the treatment regimen. It is known that great diversity exists within most solid tumors that have a common tissue of origin, like breast, and that there is even significant diversity in clinical behavior within what are described as morphologically similar tumors. Traditional approaches to breast tumor classification have utilized a mixture of empirical criteria including a morphological assessment, measures of the extent of disease dissemination, and a handful of statistically validated prognostic and predictive markers. There is a consensus, however, that these current methods fall short of the challenges posed by breast tumor diversity. The development of modern genomic analysis tools, in particular cDNA microarrays, allows us the opportunity to objectively and without foreknowledge, determine the expression level of thousands of genes in a single sample/tumor in a single day. We hypothesized that the phenotypic diversity of breast tumors would be accompanied by a corresponding diversity in gene expression patterns that we could capture using cDNA microarrays, and that this gene expression diversity could then be used to classify tumors into groups of clinical importance. We will approach the objective of defining new prognostic and predictive markers for breast cancer outcomes and response to therapy through the following specific aims:
Project 7 The transcription factor NF-kB has been shown by our group and by others to be involved with oncogenesis and to provide protection against a variety of apoptotic stimuli. Thus, we have previously shown that inhibition of NF-kB blocks cellular transformation and tumorigenesis induced by oncoproteins. Furthermore, we have shown that several chemotherapies activate NF-kB in tumor cells and that inhibition of NF-kB by expression of IkBa, the inhibitor of NF-kB, strongly enhances the cytotoxic effect of both chemotherapy and radiation via the induction of apoptosis. Based on these studies, we are examining whether the curent regimen of breast cancer chemotherapies would be augmented in their efficacy through the inhibition of NF-kB. We propose to examine breast cancer cell lines, human xenograft tumors, and breast tumors generated in animal models as to whether we can obtain enhanced responses to the current regimen of breast cancer chemotherapies when NF-kB is inhibited within the tumor. Additionally, we are proposing to examine whether other chemotherapies (such as CPT-11) which have not been effective or not tested for breast cancer will show activity against breast cancer when NF-kB is inhibited as an adjuvant approach to the chemotherapy. We will also examine whether chemotherapy treatment of breast tumors activates the expression of NF-kB-regulated genes which are known to encode proteins which block apoptosis or which provide multidrug resistance. Our studies potentially will lead to new adjuvant approaches to breast cancer therapy and will lead to the identification of NF-kB-regulated genes which may serve as markers for chemoresistance and which may ultimately serve as targets themselves for therapies to enhance the responses of breast cancer to chemotherapy. Finally, we propose a clinical trial for breast cancer utilizing adriamycin (Doxil®) in combination with PS-341, the FDA-approved proteasome inhibitor known to efficiently block NF-kB activation.
Project 8 Paclitaxel/Taxol prevents microtubule disassembly, resulting in cell cycle arrest and apoptosis. It is used clinically as a frontline therapy for ovarian tumors and has shown increasing efficacy in the treatment of breast cancer. The most encouraging data were obtained with a combination therapy using paclitaxel and anti-Her2/neu antibody. However, improvements in combination therapy with paclitaxel/Taxol for the treatment of breast cancer are still needed. Our past effort has focused on the effects of paclitaxel on gene expression, signaling and apotosis in epithelial carcinomas. We first found that paclitaxel induces the JNK/SAPK kinase system in breast carcinomas, and this activation is involved in paclitaxel-induced apotosis. In contrast paclitaxel also activates MEK1 kinase leading to an elevation of ERK1 and ERK2 activity. MEK1 members are known to be critical for cell growth and proliferation in a number of systems. By using a combination of MEK inhibitors and paclitaxel, we were able to obtain greatly enhanced tumor killing by using suboptimal levels of the two drugs. Apotosis in the presence of both drugs is five to tenfold more than with each drug alone. Additionally, we have also initiated an analysis of a derivative of epothilone B, dEpoB, which has similar effects as paclitxel. We found that dEpoB also exhibits enhanced tumor killing activity in the presence of MEK inhibitor. DEpoB has the advantage over paclitaxel in that it is not transported by the multi-drug resistant transporter system, which allows many tumors to acquire chemotherapy-resistance. Thus, dEpoB may be effective where paclitaxel is not. Used in a clinical setting, these combinations may lower toxicity and enhance tumor killing. Accordingly, the four aims of this proposal are: (1) To determine if paclitaxel and a MEK inhibitor can cause enhanced tumor elimination in a preclinical model. (2) To similarly study the combined effects of dEpoB and a MEK inhibitor. (3) To use Affymetrix analysis and microchip arrays to identify genes that are altered by the use of these drug combinations. (4) In the event that Aim (1) and/or Aim (2) show efficacy in the preclinical models, we propose to initiate a clinical trial to test the effects of combination therapy studied in Aims 1 and/or 2.
Core 1 The Molecular Epidemiology and High Throughput Genotyping Core (referred to as the Molecular Core) will provide laboratory support for Project 1 (Carolina Breast Cancer Study: DNA Repair Genes and Breast Cancer Risk), Project 2 (Carolina Breast Cancer Study: Estrogen Receptor Alterations in Breast Cancer Development) and Project 3 (Long Island Breast Cancer Study: Oxidative Stress and Breast Cancer Risk) within the Population Sciences Program, and Project 5 (Correlation of Molecular Markers with Response to Neoadjuvant Chemotherapy) within the Translational Sciences Program of the SPORE. The Molecular Core performs a wide range of PCR-based somatic genetic analyses on DNA obtained from formalin-fixed tumor sections and PCR-based genotyping on germline DNA purified from peripheral blood. In its eight years of operation, the Core has designed and implemented assays to identify gene mutations, deletions and amplification, loss of heterozygosity, microsatellite instability, complex germline variation in repetitive DNA regions such as the Hras variable number tandem repeat, and germline polymorphisms in genes involved in cellular processes such as DNA repair, carcinogen and hormone metabolism, and oxidative metabolism. The centralized nature of this facility streamlines experimental functions since the same or similar analyses may be performed for different projects. Centralization ensures highly experienced personnel, peak efficiency, stringent quality control, economic utilization of laboratory resources, technical versatility through cross training of laboratory personnel, and centralization of database and tissue tracking procedures. Furthermore, the Core has addressed the particular challenges of large-scale clinical and population-based molecular studies by designing analytical algorithms that streamline the molecular tasks and enhance reproducibility within and between data sets, developing automated approaches to increase sample throughput without loss of sensitivity or specificity, and optimizing techniques to permit analysis of the minute quantities or sub-optimal quality of DNA template obtained from paraffin-embedded tissues. In support of the proposed projects, the Molecular Core will evaluate a newly-described mutation in estrogen receptor-a, and will apply new technologic advances that increase sample throughput, including Taqman-based assays for genotyping variants in DNA repair enzymes, oxidative metabolism enzymes and the CYP19/aromatase enzyme, and the Affymetrix p53 GeneChip oligonucleotide array screening approach for detecting p53 point mutations. The Molecular Core will also assist the Tissue Procurement and Analysis Core (Core 3) in the development of immunohistochemical staining assays by identifying appropriate tissue controls based on gene expression profiles. Finally, this Core will be a resource for gene discovery efforts by UNC-CH and its collaborators in the application of newly-identified DNA repair gene polymorhpshism to the population studies of the SPORE.
Core #2: Genomics and Microarray Core The Genomics & Microarray Core provides scientific services and computational support for SPORE projects using DNA microarrays. The facility is contained within the UNC Genomics Core & Microarray Facility housed in the Lineberger Comprehensive Cancer Center. It maintains centralized equipment for microarray production, utilization, and analysis, including an Affymetrix system for oligonucleotide arrays on chips. The Core produces affordable, customized high-density cDNA microarrays on glass slides. To maximize efficiency and generate consistent quality results, the Genomics & Microarray Core will provide complete research services to SPORE projects. Investigators will isolate RNA and submit samples. The facility will prepare fluorescent probes and hybridize them to either homemade or commercially available arrays. In conjunction with the UNC Center for Bioinformatics, the Genomics & Microarray Core will provide the required computer hardware and analysis programs to collect, store, and characterize the large data sets generated by microarray research. UNC has implemented the GenoMax database and software package (Informax) to serve these computational needs. SPORE investigators will utilize this resource as well as database tools available through our collaborators at Stanford University. The Genomics & Microarray Core will support four of the SPORE projects. Its use will most likely expand to other projects and development studies as investigators recognize the advantages of this cutting edge technology.
Core 3 The SPORE Tissue Procurement and Analysis Facility (TPA) provides centralized, quality controlled, quality assured procurement, processing, analysis, storage and distribution of normal and malignant breast tissue from UNC Hospitals. Whenever possible, corresponding serum and plasma are also obtained. All procured specimens are reviewed by the Facility Pathologist to ensure that representative tissue is banked and distributed. We have recently expanded our tissue procurement activities to Rex Hospital, a large hospital in Raleigh, N.C., recently purchased by the UNC Health Care System. This effectively doubles our access to breast cancer specimens. In addition to procurement services, the facility provides tissue sectioning for frozen and paraffin embedded specimens, immunohistochemistry and fluorescence in situ assays and access to state-of-the-art tissue-based technology, including laser-capture microdissection. Expert personnel provide consultation and training to faculty and staff in these applications. The facility also serves as a central clearing house for the Carolina Breast Cancer Study -- banking and distributing unstained sections for multiple assays. Customized databases serve to monitor and survey all of these specimens, providing a coordinated system of quality control, sample tracking and distribution of specimens to appropriate investigators. The TPA has implemented policies and procedures to support these services and address technical, medical and legal issues, as well as protection of patient privacy and confidentiality. These policies developed in collaboration with area pathologists have contributed to the excellent compliance of block submission for the large population-based studies. The facility supports all of the SPORE projects and developmental studies, working with each individual investigator to accommodate their special research needs. The facility will also be the central site for tissue banking, IHC and FISH assays for the InterSPORE Neoadjuvant study.
Core 4 The Biostatistics and Bioinformatics Core provides UNC SPORE investigators with support in study design, sampling, data management, and statistical data analysis. The SPORE's statistical needs are in three primary areas: ongoing data management and analytic support for population-based research; clinical trials; and cDNA microarray studies. Dr. Qaqish, who is expert in sampling design and generalized estimating equations, will oversee the analysis of two, large population science projects (#1, #3) and contribute to ongoing data management for Cores #1 and #3. A critical component of the statistical support on these projects is taking into account sampling schemes; data management activities include maintenance user support and linkage of laboratory and epidemiologic data. Dr. Michael Schell, who is expert in clinical trials and monotone regression methods, will provide the leadership for projects #4, #5, #7 and #8. Dr. Yen-Feng Chiu, who is building a career in statistical genetics, will be the lead biostatistician for projects #2 and #6. Dr. David Fenstermacher, a bioinformatics expert and Director of the UNC Bioinformatics Group, will work with Dr. Chiu and a database programmer to support data management and analysis projects using cDNA micorarrays, primarly Project #6 (which, in turn, will inform Project #5, and other projects). The bioinformatics group will use an Oracle database as a component of the GenoMax enterprise software that will be used for many aspects of the microarray data analysis. In addition, programs such as GCG, EMBOSS, Vector NTI Suite II, Paup, and GeneSpring are available through the UNC distributed computing environment. The bioinformatics team will provide the acquisition, storage, linkage and analysis of microarray data that poses both special database and analytic needs due to the extensive volume of data that will be generated. The overall core relies on a host of design and analysis software, including SAS, S-PLUS, StatXact, nQuery Advisor, including both built-in functions and procedures and custom-designed macros and functions. Custom-designed macros that have already been developed include those for reduced monotonic regression, multiple imputation, generalized estimating equations, and the maximal chi-square test.
Core 5 The Administrative Core supports the UNC Breast Cancer SPORE's overall scientific/translational goals by providing leadership and day-to-day operations/administration. The SPORE Director leads the core. A Program Manager, an Administrative Assistant, and a Program Assistant funded from institutional sources comprise the staff. The Core organizes: the intra and inter-SPORE interactions, administrative/scientific oversight of all research projects, cores, and developmental programs; and the activities of the external, internal, and advocate advisory committees. This Core also monitors SPORE expenditures and addresses grant management issues. |
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