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Duke SPORE in Breast Cancer
Overall Abstract Director: H. Kim Lyerly, M.D. This application for continuation of the Breast Cancer Specialized Program of Research Excellence (SPORE) at Duke University proposes a highly interactive, multidisciplinary and inter-institutional program for a second period of funding. Five research projects and an ongoing developmental research project representing the translational components of five major research programs within the Duke Breast Cancer Research Program, four cores, a career development and a research development program are proposed to further our translational research in breast cancer. Project 2-1: Hypoxia and Chemoresisance in Breast Cancer, Co-Investigators- Mark Dewhirst, Larry Marks, and David Brizel. Project 2-2: T helper responses to HER2/neu in Breast Cancer Patients 2, Co-Investigators- H. Kim Lyerly, Mike Morse, and Tim Clay. Project 2-3: Genetic Modifiers of BRCA1 and BRCA2, Co-Investigators- Joellyn Schildkraut and Ed Iverson. The cores support the research programs (Core A: Administration, H. Kim Lyerly; Core B: Tissue Bank, Rex Bentley; Core C: Biostatistics and Informatics, Steve George; and Core D: Melecular and Cell Technology, Jeff Marks. The Career Development Program, chaired by O. Michael Colvin and Victoria Seewaldt, aids the development of new investigators, and the Developmental Research Program, also chaired by Dr. Seewaldt, provides funding of innovative projects. Project 1 Tumor hypoxia is related to poor prognosis in several human tumors, treated with radiation therapy, surgery and chemotherapy - alone or in combination. The reasons are multiple. Radiation and many drugs are less effective on hypoxic cells. Additionally, altered gene expression that promotes angiogenesis, cell survival and metastasis may contribute. The long-term objectives of this work are to determine whether: (1) tumor hypoxia plays a role in chemoresistance (2) improved oxygenation can lead to better outcome in the adjuvant and metastatic setting. Clinical studies and pre-clinical investigation of hypoxia modification strategies are proposed. A novel model of micrometastatic disease for both the pre angiogenic and post angiogenic phases of growth will facilitate achievement of the objectives. Hypoxia is known to induce chemoresistance to many drugs, including doxorubicin (DOX). DOX will be the exemplary drug tested, both at the clinical and pre-clinical level. Specific aim #1. We will measure tumor hypoxia in patients with locally advanced breast cancer who are treated on a Phase I-II clinical trial, testing neoadjuvant thermochemotherapy for locally advanced breast cancer. The hypothesis is that tumor hypoxia will impact negatively on tumor response from this therapy. Specific aim #2 , Using archival Breast SPORE banked tissue, from over 500 patients treated with DOX in the adjuvant setting, we will determine if hypoxia is correlated with chemoresistance. Specific aim #3. We will determine whether strategies to improve tumor PO2 (treatment with erythropoietin and/or a unique metabolic method to acutely improve tumor PO2), in the presence or absence of interference with Her-2neu (Her2) signaling, will increase DOX responsiveness in Her2 positive and negative xenograft lines. The pre-clinical model of micrometastasis allows us to serially and simultaneously monitor tumor growth, hypoxia and angiogenesis. Growth in flank tumors will also be evaluated, which simulates how treatment modifications will affect patients with metastatic disease. If positive results are obtained they would pave the way for initiation of additional human clinical trials to test hypoxia modification strategies in combination with chemotherapy.
Project 2 This project addresses the overall hypothesis that a specific T cell response of sufficient magnitude directed against tumor associated antigens (TAA) expressed by breast cancers, such as HER2/ neu , will inhibit breast cancer progression and metastasis. It has been recently shown that HER2/ neu directed immunotherapy can have clinical benefits in patients with breast cancer. Although early attempts to target HER2/ neu have focused on generating CD8+ T cell responses, often targeting the HLA A2 restricted cytotoxic T cell epitope E75. We hypothesize that we can augment CD8+ T cell responses to the E75 epitope by immunization with both E75 and class II epitopes contained in the HER2/ neu protein. This concept is supported by exciting preliminary data which demonstrate HER2/ neu specific class II restricted CD4+ T cell responses in patients using HER2/ neu intracellular domain (HER2/ neu ICD) protein loaded dendritic cells (DC). Briefly, in a pilot study of patients with metastatic breast cancer, we found that immunization with HER2/ neu ICD protein loaded DC led to a significant circulating HER2/ neu specific T helper response. Based on our data, we propose to immunize patients with mature DC loaded with both the E 75 epitope (Class I) and the HER2/ neu ICD protein (Class II) to determine the effect of cognate T help in augmenting immunity to CTL epitopes to HER2/ neu . We will determine the impact of repeated immunizations on inducing hight frequency CD+ 4 T helper cell responses. We then propose to optimize HER2/ neu loading strategies into DC by generating recombinant HER2/ neu expressing adenoviral vectors to modify DC. Finally, we will perform a clinical trial immunizing patients with HER2/ neu modified DC. This project proposes to extend our previous work with peptide pulsed DC based immunotherapy, leading to sequential clinical trials to optimize the generation of HER2/ neu specific CD8+ cytotoxic and CD4+ T helper cell responses. These clinical trials, focusing on optimizing CD4+ T helper responses to HER2/ neu , will form the background to rationally choose HER2/ neu specific vaccination strategies in trials designed to demonstrate a clinical benefit of HER2/ neu directed immunotherapy. Our specific aims are: Specific Aim 1: Perform a phase I clinical trial of HER2/neu E75 and intracellular domain (ICD) protein pulsed mature dendritic cell immunotherapy. Specific Aim 2: Monitor HER2/neu specific T cell responses using ELISPOT analysis. Specific Aim 3: Generate HER2/neu ICD encoding recombinant adenoviral vectors to optimize antigen processing ubiqunization. Specific Aim 4: Perform a phast I clinical trial of HER2/neu ICD modified dendritic cell immunotherapy. Project 3 The goal of the proposed investigation is to identify factors that influence the incidence of breast cancer in germline BRCA1/2 mutation carriers. The focus of the proposed study is to examine whether genetic factors involved in DNA damage and repair act as modifiers of BRCA1 and BRCA2. Our hypotheses are supported by findings that BRCA1 and BRCA2 are associated with protein complexes involved in various aspects of genome surveillance and repair and two recent studies reporting an interaction between a RAD51 polymorphism and BRCA2. We hypothesize that variants in the genes that encode proteins involved in DNA repair may interact with mutations in BRCA1 and BRCA2. A case-only design will be employed where cases are defined as women with breast cancer who have a known BRCA1/2 mutation status. The specific aims of this study are 1) to enroll approximately 1000 female patients diagnosed with breast cancer who have tested either positive or negative for a BRCA1 and BRCA2 mutation; 2) to assess gene-gene interactions between BRCA1/2 and polymorphisms in DNA damage and repair genes; and 3) to assess previously reported interactions between hormonally related genetic and environmental factors and BRCA1 and BRCA2 in a much larger dataset than in prior reports. Epidemiologic data and DNA will be collected for the analyses using instruments and methods developed in the context of a Cancer Genetic Network feasibility study. Women will be identified from clinic populations from 7 collaborating institutions. The molecular analyses for each of the genetic modifiers will be conducted at the Laboratory for Molecular Epidemiology by Dr. Timothy Rebbeck at the University of Pennsylvania. The Molecular and Cell Technology Core, directed by Dr. Jeffrey Marks, will perform genotyping and SNP discovery for genes involved in DNA damage and repair for which either the frequency of the polymorphism is not known or polymorphisms have yet to be identified that interact with BRCA1 and BRCA2. Innovative statistical methods will be employed to address the complex statistical issues related to examining modifiers in BRCA1 and BRCA2. The genetic epidemiology studies proposed here potentially could have profound ramifications in many areas related to the management of breast cancer including genetic counseling, prophylactic surgery, chemoprevention, and cancer therapeutics.
Project 4 In specific Aim 1 we will investigate pharmacogenomics of high-dose cyclophosphamide, cisplatin and carmustine (CPB regimen) in patients with high-risk breast cancer. This study will focus on validating our previously described relationships between polymorphisms, pharmacokinetics and outcome. In addition, we will evaluate polymorphisms which may be related to post-transplant pulmonary toxicity. Aim 2 will entail a prospective study directed at evaluation of the pharmacogenomics of standard-dose doxorubicin + cyclophosphamide (AC). An additional focus of this study will be to attempt development of a means for prediction of myelosuppression following AC therapy. In Aim 3 we will prospectively evaluate the pharmacogenomics of paclitaxel given as a weekly regimen. This study will also have an additional investigation to attempt explanation of inter-patient variability in treatment-related neurotoxicity. Justification for use of these data in a prospective fashion and identification of drug targets for future clinical studies will be provided by conduct of in vitro metabolic studies with anti-breast cancer agents suspected to be involved in polymorphic metabolism as the 4 th Aim of this Project.Within the course of this funding period, we intend to translate the work we have outlined in each of the first 3 aims into prospective clinical protocols which use a patient's genetic information to better identify the most appropriate dose and type of anticancer agent for that individual.
Core A Oversight of the Duke Breast Cancer SPORE will be maintained via a number of committees, whose administrative needs and record keeping will be maintained by the Administrative Core. In addition, specific centralized functions will be maintained in the Administrative Core, allowing for coordinated access of these functions by all SPORE investigators. The Specific Aims of the Administrative Core are: Specific Aim 1: Facilitate research opportunities within the SPORE Specific Aim 2: Monitor research progress; foster continuous improvement; set priorities Specific Aim 3: Integrate the SPORE into other interdisciplinary programs at Duke Specific Aim 4: Enhance communication and education within the SPORE, the Medical Center and the broad scientific community Specific Aim 5: Provide administrative handling of requests for use of human specimens. Specific Aim 6: Provide fiscal oversight of the SPORE Specific Aim7: Require open collaboration, resource exchange, and communication of important findings to investigators outside our SPORE Specific Aim 8: Foster participation by Patient Advocates in planning and educational activities of the SPORE Specific Aim 9: Increase participation of the Duke SPORE in inter-SPORE projects Specific Aim 10: Enhance participation of minorities in SPORE activities
Core B: Tissue Bank The Biostatistics and Information Systems Core for the Duke SPORE in Breast Cancer will support the statistical, bioinformatics and information systems needs of all projects and cores in the SPORE. The Core was developed as a collaboration between Cancer Center Biostatistics and Cancer Center Information Systems (CCIS). Dr. Stephen L. George, Ph.D., Director of Cancer Center Biostatistics and Group Statistician for the CALGB and Kimberly Johnson, Director of Cancer Center Information Systems are Co-Directors of the Core. Dr. George will direct biostatistics initiatives in this area, while Ms. Johnson will direct information systems initiatives and assist Dr. George in administering the Core. CCIS falls under the administrative oversight of Dr. George within the Cancer Center. Additional staff for this Core includes statisticians and programmers dedicated to each project and core based on their area of expertise and past associations with individual projects. In addition to the core directors, partial support is requested for three statistical personnel (1.05 FTE total) and three information systems personnel (.75 FTE total). This core includes one half-time data manager, although funding for this position will be provided by institutional funds committed to the SPORE. This Core will provide assistance in the statistical analysis of clinical, pre-clinical and laboratory studies, as well as genetic investigations. In addition, the core will support the development of research database structures and data management activities. The Core will use existing computing infrastructure resources as an efficient means to support SPORE research. Resources available to this Core include 14 servers supported by CCIS, offering over 6GB of memory and 615 GB of disk space with multi-processing capabilities and a variety of software applications and compilers.
Core C The role of the Proteomics and Emerging Technologies Core (PETC) is to provide cost effective, state-of-the-art instrumentation and techniques to the research team for the identification of proteins and protein complexes involved in the initiation, maintenance and propagation of breast cancer. The Core is directed by Dr. David B. Friedman and co-managed by Dr. David L. Hachey, with administrative oversight by Dr. Richard M. Caprioli. The PETC will offer users an array of techniques for separation of proteins obtained from tissues and cell culture by 1-D and 2-D polyacrylamide gel electrophoresis (PAGE) using high-sensitivity detection with fluorescent tags. The Core has a dedicated ion trap LC/MS instrument equipped with an automated capillary HPLC inlet. A matrix-assisted laser desorption ionization (MALDI) mass spectrometer is available for use by the PC in Dr. Caprioli's research laboratory. Additional mass spectrometry facilities are available for use on an ad hoc basis in the shared instrument center directed by Dr. Hachey. Laboratory personnel assist users in developing protein isolation and separation techniques, assist in experimental design, develop standard operating procedures, maintain QC records on instrument performance and maintenance history, perform routine assays for investigators and train students and fellows in the theoretical and practical aspects of proteomics. The Core is run as a limited-access facility in which users are expected to prepare their own samples and subsequently bring them to the PC for separation and analysis. The Core has dedicated operators for protein separations and mass spectrometric analysis who are available to assist users as necessary. Administrative staff monitors the use of the instrument facilities by investigators and prepares reports on utilization for the SPORE director. The PI's of Project 1, 2 and 3 have requested proteomics services. The most frequently requested services are those related to identification and characterization of proteomes by 2-D PAGE and MS, analysis of protein expression by MALDI profiling, and protein expression using multispectral analysis of differentially labeled proteins.
Core D The Molecular and Cell Technology Core is an outgrowth of the previously described Tissue and Cell Technology Core. In our current configuration, tissues collection and pathology expertise will be administered from the Tissue Bank (Core B) while this Core will provide storage, databasing, and technology services associated with these specimens. In addition, specialized xenograft technology will also be run from this Core. Technology, in the form of macromolecular extraction and genotyping will specifically service two of the projects. Finally, a wide range of resources, technology, information, and services are provided to SPORE and other Duke investigators working on breast cancer. This Core will establish a close interface with the Tissue Bank Core to maintain existing tissue and blood resources, store newly collected breast specimens, and assemble pathologic and clinical data associated with these specimens. The core has the following four aims: 1) Storage and maintenance of the tissue and blood specimens from breast patients will continue to be handled by this facility. Detailed patient and tumor information will be captured in relational databases created for this resource. 2) A series of innovative animal models and xenograft cell lines for use in these animals have been developed within this Core and will be provided to support Project 2-1. 3) Histopathology support will be provided including immunohistochemistry and laser capture microdissection fully backed by dedicated pathologists. Macromolecular extraction of blood, tissues and cells will be handled through this facility to support Projects 2-3 and 2-4. 4) This Core will provide high-throughput genotyping services to Projects 2-3 and 2-4 to support their research. SNP discovery and population frequency estimates will also be performed. The strength of this Core is the combined expertise and resources of the Core leaders. |
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