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Johns Hopkins SPORE in Breast Cancer Nancy E. Davidson, M.D.
Project 1 - Molecular Markers for Breast Cancer Each year about 50,000 new cases of ductal carcinoma in situ (DCIS) are diagnosed in the US, and account for 17-34% of all mammographically detected cases. Disease recurs within 10 years in 20% of women treated for DCIS. Currently, no clinical or histopathological characteristics have been found that are consistently associated with recurrence. Promoter hypermethylation is a powerful epigenetic mechanism for suppression of gene expression and occurs in many types of cancers. Hypermethylated genes provide very sensitive and specific DNA markers for breast cancer. A quantitative method [Quantitative Multiplex-Methylation Specific PCR (QM-MSP)] was developed in the investigator’s laboratory, which detects hypermethylation in multiple genes in very small biological samples of cancer cells in the presence of a vast excess of normal cells, with high sensitivity and specificity. This proposal is based upon the hypothesis that the hypermethylation of a specific set of genes in DCIS can be used to recognize those DCIS cases that will recur as invasive cancer. The goal of this proposal is to identify genes that are methylated in DCIS, study their behavior during tumor progression, and develop a recurrence model based on a DCIS-specific gene methylation panel to test their utility as predictors of tumor recurrence in patients with long-term follow-up This goal will be achieved in three steps: In Aim 1, perform a genome-wide scan for genes silenced by hypermethylation using a modified array analysis on purified epithelial cells, isolated from 30 primary DCIS and treated with demethylating agents. In Aim 2, determine if the genes identified in Aim 1 support a model of progression from DCIS to invasive breast cancer. The genes, following verification, will be used for QM-MSP analysis of archival normal, benign, DCIS and invasive cancer samples, and in analogous samples obtained from 44 individual mastectomy specimens. In Aim 3, test the ability of the methylated gene marker panel derived in Aim 2 to prognosticate recurrence of DCIS as invasive ductal carcinoma, and thereby identify patients at risk. The most promising gene panel will be tested in training and test sets by QM-MSP in tumor samples from UCSF’s DCIS case-control study. In this cohort of 1062 women treated only by lumpectomy, 200 cases recurred within 7 years, and are matched with 400 age-, grade-, size- and margin-matched control cases that did not recur. As a result of this work, we hope to derive an algorithm that will identify DCIS patients at risk for recurrence, and derive a molecular progression paradigm that will permit the development of a therapeutic strategy that will improve tumor-free survival among those patients, while minimizing excessive therapy in those unlikely to develop invasive disease.
Project 2- Exploiting Epigenetic Alterations in Breast Cancer Inactivation of genes that regulate cell proliferation and death is a critical part of the neoplastic process. Crucial genes like tumor suppressor genes can be inactivated via gene deletion, point mutation, or inhibition of transcription. Transcriptional silencing through epigenetic mechanisms is associated with acquisition of promoter methylation and changes in chromatin structure through histone modification leading to a repressive chromatin state. Attempts to relieve this transcriptional repression have led to consideration of clinical trials using inhibitors of DNA methyltransferases (DNMT) and histone deacetylases (HDAC) in cancer. Our previous studies suggest that DNMT or HDAC inhibitors or the combination can reactivate expression of several epigenetically silenced genes in breast cancer, including the estrogen receptor alpha (ER) in human breast cancer cells. This proposal will build on these observations and the recent availability of agents that can be administered clinically to address the hypothesis that targeting epigenetic mechanisms with a single agent or combination approaches will be an effective strategy for breast cancer treatment through three specific aims: Specific Aim 1: Complete a clinical trial to determine the biological effects of an oral HDAC inhibitor, SAHA, in women who are undergoing primary surgery for breast cancer; Specific Aim 2: Optimize dose and schedule of potential combination therapies in cell culture and xenograft models using clinically relevant agents, and Specific Aim 3: Use the findings from the preoperative clinical trial of SAHA in Specific Aim 1 and the preclinical models in Specific Aim 2 to design a rational trial of combination therapy for women with breast cancer. Because of compelling preclinical data that HDAC inhibitors reactivate expression of ER in ER-negative human breast cancer cell lines and thereby sensitize these cells to growth inhibition by tamoxifen, the primary focus will be on a strategy of tamoxifen and HDAC inhibitor. The studies in this renewal application will continue to enhance our understanding of epigenetic mechanisms as potential therapeutic targets, establish the biological activity of one HDAC inhibitor in women with breast cancer, and lay the foundation for rational clinical trials.
Project 3- Combinatorial Vaccine Approaches for the Treatment of Breast Cancer The broad objective of this proposal is to identify combinatorial vaccine approaches that will result in effective immunization against breast cancer. Immune tolerance mechanisms (systemic and at the tumor site) provide formidable barriers to effective immunization in cancer patients. HER-2/neu transgenic (neu-N) mice demonstrate immune tolerance to the endogenously expressed rat HER-2/neu (neu) protein. We have successfully used these mice to define systemic tolerance mechanisms and to identify combinatorial vaccine approaches that can overcome them. We recently identified RNEU420-429 as the immunodominant MHC I epitope in rat neu. RNEU420-429-specific T cells isolated from vaccinated neu-N mice exhibit lower avidity than T cells isolated from the parental non-tolerized mice (FVB/N). Immune modulating doses of Cyclophosphamide (Cy) in sequence with vaccine in neu-N mice produce RNEU420-429-specific T cells that have 10-fold higher avidity as compared to T cells from neu-N mice given vaccine alone. The enhanced avidity of the T cells correlates with the mouse’s ability to reject neu-expressing mammary tumors. Modulation of additional mechanisms of systemic T cell tolerance together with the modulation of mechanisms of immune tolerance in the tumor’s microenvironment in these mice, should lead to the development of new and improved combinatorial immunotherapy strategies for the treatment of breast cancer patients. In aim 1, we will evaluate immune modulating agents that will effectively activate neu-specific CD8+ T cell populations specific for the non-dominant neu epitopes. In aim 2, we will evaluate immune modulating agents that will effectively activate low avidity neu-specific CD8+ T cell populations specific for the RNEU420-429 epitope. In aim 3, we will evaluate pathways within the tumor’s microenvironment (two new B7 family members) that are barriers to effective tumor eradication in vivo. In aim 4, we will evaluate the frequency of expression of these tolerizing signals by human breast cancer specimens. In aim 5, we will determine the therapeutic value of the most potent immune modulating agent/vaccine combinations in treating spontaneously arising neu-expressing mammary tumors and liver metastases. The most effective combinations identified in aims 1-4, will be tested in patients with breast cancer. However, funding for the clinical trials that arise from this grant will come from other sources.
Project 4- Chemopreventive Efficacy of Broccoli Sprouts Against Breast Cancer Epidemiological studies provide convincing evidence for an inverse relation between quantities of fruit and vegetables consumed and the risk of developing cancer. Cruciferous vegetable (e.g., broccoli) consumption has been recently associated with reduced risk for breast cancer. A major mechanism for protection against carcinogenesis involves induction of phase 2 detoxication enzymes that promote elimination of carcinogens and boost antioxidant capacity. Many edible plants, most notably 3-day-old broccoli sprouts, contain potent phase 2 enzyme inducer activity in the form of isothiocyanates or their glucosinolate precursors. In animals, sulforaphane, derived from the principal glucosinolate (glucoraphanin) of broccoli sprouts, is a very potent inducer of phase 2 enzyme activity and protects against chemical carcinogenesis. The objective of this study is to translate and evaluate these laboratory findings in a human population at elevated risk for breast cancer. Previous trials have afforded important information on the safety, metabolism, and urinary disposition of isothiocyanates, and on the activation of glucosinolates. Therefore, our aims are to 1) determine the pharmacodynamic action of a broccoli sprouts preparation, as well as pure sulforaphane and pure glucoraphanin, in the mammary epithelium of rodents to develop better biomarkers; 2) evaluate the pharmacokinetics and pharmacodynamic action of broccoli sprouts in 30 healthy women undergoing breast reduction surgery or prophylactic mastectomy by conducting a 10-day randomized placebo controlled clinical trial that measures isothiocyanate levels in blood, urine and mammary tissue as well as phase 2 inducer activity, mRNA transcript levels and estrogen metabolites in mammary tissue; and 3) test whether a 3 month intervention of a broccoli sprout preparation modulates a panel of breast cancer risk biomarkers (Ki67 expression, estrogen metabolite profiles, novel markers) compared to placebo in 126 women at increased risk for breast cancer using a Phase II randomized clinical trial design. These studies will provide a rigorous assessment of the usefulness of modulating expression of carcinogen detoxication enzymes and other sulforaphane target genes by means of a food containing a standardized level of a phytochemical as a general strategy for chemoprevention in humans. Core 1- Administration Core The Administration Core will continue to be responsible for managerial oversight of all Johns Hopkins Breast Cancer SPORE Program activities. In addition the Administration Core will help facilitate interactions between Johns Hopkins Breast Cancer SPORE Program investigators and investigators associated with other Breast Cancer SPOREs. It will continue to assist in the orchestration of productive responses to new National Cancer Institute initiatives such as the NCI-Avon Progress for Patients Program. The structure of the Johns Hopkins Breast Cancer SPORE with its Principal Investigator, Research Project and Core Co-Leader Committee, Steering Committee, Institutional Advisory Board, and External Advisory Board has been designed to promote translational research by creating a breast cancer research culture that transcends academic departments, medical disciplines, and individual research skills. It provides high quality monitoring, evaluation, and oversight of all aspects of the Breast Cancer SPORE portfolio of Research Projects, Core Resources, Career Development Program, and Developmental Research Program and NCI Avon Progress for Patients Awards in a low-cost and efficient administrative structure. This highly experienced team facilitates all communications and meetings, meets all National Cancer Institute reporting requirements, maintains adherence to SPORE Program budget constraints and requirements, ensures compliance with NIH and Hopkins research guidelines, coordinates attendance of SPORE Program investigators at various SPORE-associated meetings, participates in SPORE Principal Investigators conference calls, and organizes responses to supplemental funding opportunities. Core 2- Human Breast Tissue and Pathology Core The Human Breast Tissue and Pathology Core (HBTPC) resource provides essential services to all projects in the Johns Hopkins Breast Cancer SPORE in three major areas: tissue acquisition and processing, tissue histology and analysis, and pathologist consultation. As an integral component of our Breast Cancer Program, this Core also provides these services in collaboration with investigators of other SPORE programs, other breast cancer research programs at Johns Hopkins’ Sidney Kimmel Comprehensive Cancer Center (SKCCC), and a number of collaborating investigators at other institutions. It is therefore a unique and essential resource. For tissue acquisition and processing, the HBTPC oversees the collection and distribution of fresh and fresh-frozen specimens, including samples of invasive carcinoma, in situ carcinoma, metastatic carcinoma, and normal breast from breast cancer patients and from healthy (breast reduction) patients. In addition to prospective collection, the HBTBC has reviewed and catalogued similar specimens that had been collected prior to the SPORE funding and stored in the Pathology Department tissue bank, providing a resource of over 1200 frozen samples. The HBTPC continuously addresses needs of specific projects in the Breast SPORE program, by initiating collection and distribution of peripheral blood and duct lavage specimens, establishing explants for ex vivo testing of novel therapeutic agents, constructing tissue arrays that represent the full spectrum of neoplasia of the breast, and overseeing an immediate autopsy program that harvests tissues from widely metastatic, advanced cancers. For tissue histology, the HBPTC utilizes the existing Reference Histology and Immunohistochemistry laboratories in the Department of Pathology. The Core also uses a tissue microarray laboratory in Pathology and laser microdissection equipment in the Cancer Center, providing these services in an integrated, cost-effective manner. Perhaps the most important contribution of this Core to the Program is that of expert pathologist consultative services. Two pathologists, experienced in both the disciplines of surgical pathology and breast cancer research, work closely with SPORE investigators in experimental design, use of human tissues, and interpretation of data involving analysis of human tissues. These investigators (Drs. Argani and Gabrielson) have co-authored 43 breast cancer publications during the five years of SPORE funding, reflecting their engagement in the program and the contribution of this core to breast cancer research at Johns Hopkins. Core 3- Biostatistics and Bioinformatics Core This Core resource will provide comprehensive biostatistics and bioinformatics consultation and collaboration to all projects in the proposed Johns Hopkins Breast SPORE. In addition, it will provide support for data storage, informatics, and computing, and assist with the identification and solution of complex data tasks arising in the course of project activities. Core members will work with project investigators across a wide spectrum of activities, encompassing data acquisition (including study design, feasibility of objectives, availability of public-access genomic information, and data storage), statistical quality control (including artifact detection and preprocessing of data from genomic technologies), data analysis (including visualization, biostatistical modeling, and assistance with manuscript writing), and development of innovative customized biostatistics and bioinformatics methodologies and tools if required by specific projects. The Breast SPORE Biostatistics and Bioinformatics Core is housed in the Division of Clinical Trials and Biometry of the Department of Oncology, an active and committed group of biostatistics and bioinformatics faculty members, with access to state-of-the art equipment and a broad range of expertise. This Core resource is the continuation of an existing resource within the original and current Breast SPORE program at Johns Hopkins. Core members have a strong commitment to this SPORE, stemming from: a) a history of collaboration with the investigators of this as well as other SPORE projects b) an active and independently funded agenda of synergistic progects in cancer genetics and genomics, with substantial breast cancer components and c) a demonstrated interest and understanding of both the biological and analytical questions and challenges. All proposed projects are anticipated to make use of this resource in every aim.
List of Investigators involved in SPORE Projects Project 1 Sukumar, Sara Wolff, Antonio Argani, Pedram Fackler, Mary Jo Umbricht, Christopher Baylin, Stephen Herman, James Garrett-Mayer, Elizabeth Project 2 Davidson, Nancy Stearns, Vered Sukumar, Sara Carducci, Michael Altiok, Soner Rudek, Michelle Parmigiani, Giovanni Garrett-Mayer, Elizabeth Project 3 Jaffee, Elizabeth Emens, Leisha Chen, Lieping Pardoll, Drew Armstrong, Todd Parmigiani, Giovanni Project 4 Kensler, Thomas Visvanathan, Kala Stearns, Vered Talalay, Paul Yager, James Fahey, Jed Dolan, Patrick Garrett-Mayer, Elizabeth Core 2 Argani, Pedram Gabrielson, Edward Core 3 Parmigiani, Giovanni Garrett-Mayer, Elizabeth Zahurak, Marianna Zhang, Zhe |
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