|
|
||||
|
|
|
|
|
 |
 |
 |
 |
 |
 |
 |
|
|
Organization of the Organ Systems Branch Current SPORE Programs Information for the Public PART Program Information for Applicants IntraSPORE Communications
|
DANA-FARBER CANCER INSTITUTE
OVERALL ABSTRACT This application creates a Specialized Program of Research Excellence (SPORE) in Breast Cancer at Harvard University and within the newly configured Dana-Farber/Harvard Cancer Center. The Harvard SPORE includes investigators from all Harvard-affiliated hospitals in Boston and from the Harvard Medical School and School of Public Health. Our SPORE will translate findings from the epidemiology of breast cancer into a novel clinical trial, seeking to improve risk assessment and test a preventive intervention. Investigators seek new susceptibility genes and will test their effects in at-risk and affected women. We will attempt to apply fundamental discoveries about the function of BRCA1 and BRCA2 in the assessment of women, who may carry gene mutations in these or similar genes. These investigations will be integrated by creation of a common repository of high-risk women and their families, which includes all Harvard hospitals and clinics. New genes will be sought by a genetic approach in early-stage breast cancer. This approach will be complemented by a functional screen for genes active in assays of breast cancer progression. These genes, and their pathways, will be used to screen combinatorial libraries for small-molecule inhibitors. Animal models of critical events in breast cancer progression will be sought by transgenic and knockout technology. These studies will be integrated by collaboration, exchange of reagents and use of shared tissues and pathology services. In-situ cancer is increasingly common in clinical practice. Our SPORE will record the perceptions of women with newly diagnosed intraductal cancer, drawing on large populations and rapid case ascertainment. Women with progressive stages of breast cancer will be examined to map the development of immune dysfunction so that we can design rational interventions. The last two projects concern women with diagnosed breast cancer. All clinical-translational research will be integrated by common clinical databases, data management and statistical analysis. The Harvard SPORE will devote more than 10% of its direct research dollars to develop new projects and cores, and to support the careers of new translational scientists. Several strategies will provide a flexible and changing research agenda.
PROJECT 1 A large body of epideiologic evidence has consistently linked reproductive factors to breast cancer. Ages at menarche and first pregnancy are parameters in established breast cancer risk models. Obesity and hormone replacement therapy also increase postmenopausal breast cancer risk. SERMS, such as tamoxifen and raloxifene, selectively inhibit estrogen at the receptor, and reduce breast cancer risk. Despite its efficacy, however, tamoxifen is not being widely prescribed to reduce breast cancer risk. This is due to perceptions about side effects and the inability to identify women who most benefit from tamoxifen. We and others have shown that postmenopausal levels of serum estrogens are highly correlated with breast cancer risk. Strategies of estrogen deprivation are increasingly prominent among the various therapeutic hormonal interventions used for hormone receptor positive tumors. In premenopausal women, adjuvant chemical or surgical oophorectomy may improve survival. In postmenopausal women, agents that inhibit aromatase, the critical enzyme in conversion of precursors to estrogen reduce estrogen levels in the circulation, breast tissue, and tumors. Currently available aromatase inhibitors are highly selective and substantially reduce circulating estrogen levels without altering other aspects of steroid production. They are effective in the treatment of metastatic disease, and a large adjuvant trial is in progress. The goal of this project is to improve breast cancer prevention by translating these observations into clinical practice. In aim one, we will test the hypothesis that by incorporating data on serum estrogen levels and mammographic density from the Nurses' Health Study we can improve current models of individual breast cancer risk. In the second aim, we will develop a novel paradigm for breast cacer prevention among postmenopsual wmoen at increased risk based on high levels of circulating estrogens. We will access whether an aromatase inhibitor is sufficiently well tolerated (by its effects on bode density, menopausal symptoms and blood lipids) to permit further development for chemoprevention. If successful, these studies will contribute to the adoption of a quantitative paradigm of breast cancer risk estimation and reduction that more closely parallels the widely accepted models of cholesterol and blood pressure titration that have transformed cardiovascular disease in the US.
PROJECT 2 Germline mutations in TP53 account for ~70% of cases of Li-Fraumeni Syndrome (LFS), a familial cancer phenotype associated with breast cancer, sarcomas, and other tumors. We hypothesized that LFS kindreds without TP53 mutations might harbor mutations in other components of this DNA damage response pathway, and we have recently shown that a subset of these cases do have germline mutations in hCHK2, the mammalian homolog of a kinase that regulates the G2 checkpoint in yeast. These observations raise the possibility that other genes in the G2 checkpoint may contriute to genetic predispostion to breast cancer. Furthermore, the development of early-onset and bilateral breast cacner in kindreds with hCHK2 mutations suggests that alterations in this gene may occur in women who have evidence of predisposition to breast cancer but who lack the classical manifestations of LFS. We propose three major aims: 1. Mutational analysis of G2 checkpoint genes in a highly selected cohort of patients who have breast cancer in the context of multi-cancer syndromes without germline mutations in p53. The absence of p53 mutations in such families enhances the likelihood of detecting mutations in related genes required for genomic stability. 2. Mutational analysis of hCHK2 and related genes in subsets of the general population at increased risk for breast cancer, but without the extraordinary risk factors demonstrated by LFS kindreds. These patients may have mutations with lower penetrance, associated with attenuated cancer phenotypes. 3. Mutational analysis of these genes in breast cancer specimens, to determine their contribution to the development of sporadic breast cancer. Together, these studies will address the contribution of a new class of tumor suppressor genes in both familial and sporadic cases of breast cancer.
PROJECT 3 This proposal aims to apply recent advances in the understanding of BRCA1 and BRCA2 function to the clinical problem of uncharacterized variant alleles of BRCA1 and BRCA2. As more women have their BRCA1 and BRCA2 genes sequenced, distinguishing between true disease-causing alleles and benign polymorphisms has become a critical clinical issue. At present, no assay exists that reflects the tumor suppression activity of these genes. In Specific Aim 1 we propose to develop an assay based on the ability of various alleles to complement defects in radiation resistance and DNA double strand break repair in a BRCA1-deficient human tumor cell line. In Specific Aim 2, we propose to explore the possibility that BRCA1 and BRCA2 demonstrate haploinsufficiency. If this is the case, we will investigate the possibility that any such phenotype could form the basis for an assay aimed at distinguishing benign from disease-linked alleles of BRCA1 and BRCA2.
PROJECT 4 Ductal carcinoma in situ (DCIS) of the breast is a heterogeneous disease with varying clinical outcomes. Current classification of DCIS is based on cytological and architectural features. Although some of these features correlate with the risk of local recurrence, they can be determined only with moderate consistency and none of them is able to accurately predict the development of invasive disease. Therefore the identification of novel molecular markers that correlate with the clinical behavior of DCIS is of utmost importance. The specific goals of this proposal are aimed at the identification and evaluation of such markers using the combination of comprehensive gene expression analysis and tissue microarrays. In order to define genes implicated in DCIS, we have analyzed the gene expression profiles of DCIS and normal mammary epithelium using SAGE (Serial Analysis of Gene Expression) and identified over 300 differentially expressed transcripts. To further analyze how reproducibly these genes are differentially expressed we propose to generate tissue microarrays from hundreds of samples of benign breast tissue and in situ and invasive breast carcinomas. These tissue microarrays will then be used to rapidly screen DCIS specific transcripts using in situ hybridization and immunohistochemistry. The clinical usefulness of genes with consistently different expression in DCIS will be evaluated using a tissue microarray generated from DCIS cases with known clinical outcome. Identification of clinically useful molecular markers that would predict the behavior of DCIS could lead to the individualized treatment of patients diagnosed with this disease.
PROJECT 5 The overall objective of this proposal is to identify proteins or protein pathways that mediate critical aspects of mammary tumor progression and to develop screens for small molecule inhibitors of these pathways. This will be accomplished using a functional genomics approach that involves infecting mammary epithelial cells with libraries of retroviruses encoding candidate tumor progression genes and screening for cells that display various properties of tumor cells in culture. The genes responsible for these phenotypic alterations will then be identified and cloned. We will then validate these genes as potential therapeutic targets based on whether these genes are altered in sequence or expression in breast tumors. Once validated targets are identified, we will then collaborate with the Institute of Chemistry and Cell Biology (ICCB) and industry to identify candidate small molecule modulators of these critical targets. The functional assays developed for this project provide the means to screen though large numbers of genes from breast tumor cells in order to identify genes that are able to mediate phenotypic alterations associated with tumor progression. The assays will be used in screens to identify novel genes from breast tumor cells, in establishing which properties of mammary tumor cells are regulated by known genes associated with breast cancer and in determining whether genes identified though high throughput expression analysis of breast tumor cells can mediate any phenotypic alterations in mammary cells. Collaborations with other SPORE projects will be critical in providing genes for analysis and for analyzing tumor samples to evaluate the relevance of the genes to human breast cancer. Lastly, through collaborations with the ICCB and industry, we plan to translate these findings into meaningful interventional strategies through the development and execution of screens for small molecule inhibitors that will interfere with breast cancer progression.
PROJECT 6 The rapid growth of enetic insights into causes of breast cancer continues to identify new targets for gene-specific therapies. A screen predicting their potential efficacy is needed. Transgeneic mice have been underutilized for this purpose, considering their unique ability to mimic gene-specific effects in vivo. Cyclin D1 is amplified in 15 percent of breast cancers and overexpressed in nearly 40 percent. Its expression identifies estrogen-positive breast cacners with a poor prognosis. It uniquely drives abnormal proliferation all the way to frank malignancy when expressed in transgenic mammary glands, unlike any other tissue. The mammary gland is a particular target of cyclin D1 loss in knockout studies. Reagents developed in our laboratories include MMTV-cyclin D1 mice, cyclin D1 knockouts, and a new MMTV-p16 strain. Using our mouse strains, additional MMTV-oncogene transgenic strains, p16 peptide fusions with TAT (or Antennapedia) protein transduction domains, and drugs that inhibit cyclin dependent kinase function, we seek to validate the use of transgenic models as a pre-clinical test system. We propose to identify genetic pathways where intact cyclin D1 activity is needed for tumorigenesis. 1. The phenotype of crosses between cyclin D1 knockout mice and transgenic mice targeting expressions of c-myc, ras, growth factor- (TGF ), Wnt-1, Py T antigen (src pathway) and Her2/Neu to mammary epithelium will be examined. The role of cyclin dependent kinases in any protective effects will be studied using additional crosses between MMTV-p16 mice and transgenic mice expressing the same set of genes. 2. To evaluate the effects of blocking cyclin D1 in pre-neoplastic lesions versus fully transformed lesions we will develop regulated excision of the cyclin D1 locus and evaluate loss of cyclin D1 function in spontaneously arising tumors. To extend this test to cdk function, we will also develop tetracycline-regulated, tissue-specific targeting of p16INK4A. Using p53 -/- xMMTV-cyclin D1 mice, we will expand the preneoplastic cyclin D1-expressing population by transplanting mammary glands into wide type recipients. 3. We will evaluate the relevance of these genetic manipulations by comparing their effects to the results of treatment using small molecular antagonists of cyclin D1/cdk. Specifically we will test protein transduction with Anennapedia or TAT-p16 peptide fusions as gene therapy in vivo. We will follow these studies by evaluating the therapeutic efficacy of chemical inhibitors of Cdks in the treatment of tumors caused by transgenic over-expression of oncogenes in mammary tissues.
PROJECT 7 The rapid growth of genetic insights into causes of breast cancer continues to identify new targets for gene-specific therapies. A screen predicting their potential efficacy is needed. Transgenic mice have been under-utilized for this purpose, considering their unique ability to mimic gene-specific effects in vivo. Cyclin D1 is amplified in 15 percent of breast cancers and overexpressed in nearly 40 percent. Its expression identifies estrogen-positive breast cancers with a poor prognosis. It uniquely drives abnormal proliferation all the way to frank malignancy when expressed in transgenic mammary glands, unlike any other tissue. The mammary gland is a particular target of cyclin D1 loss in knockout studies. Reagents developed in our laboratories include MMTV-cyclin D1 mice, cyclin D1 knockouts, and a new MMTV-p16 strain. Using our mouse strains, additional MMTV-oncogene transgenic strains, p16 peptide fusions with TAT (or Antennapedia) protein transduction domains, and drugs that inhibit cyclin dependent kinase function, we seek to validate the use of transgenic models as a pre-clinical test system. We propose to identify genetic pathways where intact cyclin D1 activity is needed for tumorigenesis. 1. The phenotype of crosses between cyclin D1 knockout mice and transgenic mice targeting expression of c-myc, ras, transforming growth factor-a (TGFa), Wnt-1, Py T antigen (src pathway) and Her2/Neu to mammary epithelium will be examined. The role of cyclin dependent kinases in any protective effects will be studied using additional crosses between MMTV-p16 mice and transgenic mice expressing the same set of genes. 2. To evaluate the effects of blocking cyclin D1 in pre-neoplastic lesions versus fully transformed lesions we will develop regulated excision of the cyclin D1 locus and evaluate loss of cyclin D1 function in spontaneously arising tumors. To extend this test to cdk function, we will also develop tetracycline-regulated, tissue-specific targeting of p16INK4A. Using p53-/- x MMTV-cyclin D1 mice, we will expand the preneoplastic cyclin D1-expressing population by transplanting mammary glands into wild type recipients. 3. Finally, we will evaluate the relevance of these genetic manipulations by comparing their effects to the results of treatment using small molecule antagonists of cyclin D1/cdk. Specifically we will test protein transduction with Antennapedia or TAT-p16 peptide fusions as gene therapy in vivo. We will follow these studies by evaluating the therapeutic efficacy of chemical inhibitors of Cdks in the treatment of tumors caused by transgenic over-expression of oncogenes in mammary tissues.
CORE 1 Women differ in their risk of developing breast cancer. Studies of individuals and families with exceptionally high risk have led to the identification of inherited breast cancer susceptibility genes, including BRCA1, BRCA2, p53, and recently, chk2. These and other cancer-predisposing genes were identified primarily through studies of unusual patients with multiple primary cancers, early-onset cancers, precancerous lesions or cancers associated with malformation syndromes. Within this SPORE, the High Risk Core will identify patients at high risk of breast cancer who are likely to be informative in future cancer research. Our Core recruitment criteria include: families carrying one of the high penetrance breast cancer predisposing genes; early onset breast cancer particularly with a with a family history of either breast or ovarian cancer; family cluster of three or more first-and second-degree relatives with either breast or ovarian cancer; breast cancer diagnosed before age 45 with family history of childhood cancers; breast cancer under 45, associated with major malformations or another primary cancer; or precancerous lesions (ADH, DCIS or LCIS) at any age. Our Core staff will consent eligible individuals and families to respond to a standard questionnaire and donate blood samples and their fresh or fixed tumor tissues for research. All tissue samples will be delivered to the Tissue and Pathology Core under Drs. Harris and Schnitt for processing, storage and future distribution. After replacing personal identifiers with codes, data on biospecimen availability and questionnaire information will be entered into a searchable database that can be accessed by approved SPORE investigators. Blood sample and tumor specimens will also be made available with approval. The High Risk Core will also facilitate re-contact of patients and families for additional collection of specimens and data, including mortality and the occurrences of new cancers on follow-up. Based on the proposed personnel, we will enroll at least 1,000 breast cancer cases over the next 5 years and at least 4,000 of their affected and at-risk relatives (total 5,000 family members).
CORE 2 The purpose of the SPORE Tissue and Pathology Core is two-fold: 1. to provide research pathology services to SPORE investigators, including histology, immunohistochemistry, in situ hybridization, computer-assisted image analysis, digital imaging, laser capture microdissection, tissue microarrays, rodent pathology services, and immortalization of cell lines; and 2. to provide a tissue/blood repository for use by SPORE investigators, including collection, freezing, and storage of fresh samples of breast cancer and benign breast tissue, and collection, processing, and storage of blood and blood components (plasma, peripheral blood mononuclear cells, and circulating tumor cells) from patients enrolled in SPORE-related projects. In addition a virtual specimen bank will be created which will link together the extensive existing tissue resources among the Dana Farber/Harvard Cancer Center hospitals. All SPORE investigators will have access to the material in these banks. The research pathology services are essential for the evaluation of new markers, probes and antibodies developed by SPORE investigators. The tissue/blood repository will provide SPORE investigators with access to a wide range of clinical material with associated information. In this regard, there will be close collaboration between the Tissue and Pathology Core and the Clinical Database and Data Management Core which will be responsible for providing and maintaining the clinical databases for samples stored in the tissue/blood repository. Thus, one of the major goals of this core facility is to create a tissue and blood resource, linked to clinical data, behind a secure data management system that will be available to DF/HCC SPORE investigators as well as SPORE investigators at other institutions.
CORE 3 This core provides consultation and collaboration on quantitative methods to investigators on all SPORE projects and developmental projects, and other cores. The specific aims are: 1. To provide "open door" (more likely "open phone") short term statistical consultation to the entire group of SPORE investigators. 2. To provide biostatistical expertise for the planning, conduct, analysis, and reporting of laboratory, animal, clinical, and epidemiological studies. 3. To provide consultation on computer databases, statistical computer packages, publicly available statistical programs and moving data between computers and between databases, as well was to provide statistical collaboration in developing statistical programs for specialized problems.
CORE 4 The purpose of the Clinical Data and Data Management Core (CDDM) is to support the clinical data requirements of current and future SPORE projects. Three distinct activities are encompassed within this core: (1) data management for clinical trials conducted through the SPORE; (2) collection of clinical data on patients with breast cancer receiving their care within SPORE institutions; and (3) maintenance of the database storing information on specimens processed by the Tissue and Pathology Core. Many of the SPORE projects will require the services provided by the CDDM Core. For projects involving a clinical trial, data managers of the CDDM will perform all tasks related to completion of study forms for trial participants. The Clinical Research Information System (CRIS) a multi-institutional relational clinical database will support SPORE activities in three ways. First, survey data from patients will help identify families for possible inclusion in the High-Risk Patients and Families core, or subjects eligible for specific studies. Second, CRIS data linked to specimens will allow for correlations between specific laboratory findings and clinical outcomes. Third, CRIS will serve as the database for selected observational studies. The Specimen Tracking Information Program (STIP) will support the handling of patient specimens for a number of SPORE projects, and is instrumental in constructing the multi-institutional virtual SPORE specimen bank. The activities of the CDDM Core will facilitate current and future translational research studies. Working together, the HRPF, TP, and CDDM Cores will generate a very large bank of specimens linked to comprehensive coded clinical data on risk factors, stage at diagnosis, response to therapy, complications, and survival. Studies using this resource to examine the relationship between established and experimental laboratory assays and risk factors/outcomes have the potential to make important contributions to our understanding of the etiology, prevention,and treatment of breast cancer. |
 |
|||
|
|