|
|
||||
|
|
|
|
|
 |
 |
 |
 |
 |
 |
 |
 |
|
| Brigham and Women's Hospital
Overall Abstract In this proposal, we seek to establish a Specialized Program of Research Excellence (SPORE) in Skin Cancer at Harvard University and Brigham and Women's Hospital, within the newly configured Dana-Farber Harvard Cancer Center. The Dana-Farber Harvard Cancer Center SPORE includes investigators from Harvard Medical School and School of Public Health, as well as the Brigham and Women's Hospital, Beth Israel Deaconess Medical Center, Children's Hospital Medical Center, Dana-Farber Cancer Institute, and the Massachusetts General Hospital. These institutions have come together around the theme of translational research in melanoma and cutaneous oncology. Five Projects, five Shared Resources (Cores), a Developmental Program, and a Career Development Program are proposed. Project 1 will translate epidemiologic findings into the development of improved risk models for melanoma; once developed, these will be validated across a large patient population. Project 2 employs the power of whole genome transcriptional profiling with the ultimate goal of improving our ability to diagnose and predict the biological course of primary melanoma. Project 3 tests the hypothesis that cutaneous T cell lymphoma is a malignancy of a population of memory T cells that normally performs immunosurveillance of the skin, and seeks to identify new targets for therapy. Projects 4 and 5 are exciting translational clinical trials. In Project 4, two diametrically different dendritic cell vaccination strategies are directly compared, using novel immunological endpoints. In Project 5, biochemotherapeutic approaches are employed to treat metastatic melanoma. Immunologic monitoring will be performed, again with the goal of improving therapy for melanoma patients. These five Projects are integrated by five Cores. The Biostatistics Core provides expert consultation to each study, while the Tissue and Pathology Core provides controlled and organized access to tissue vital for these projects. An Immune Monitoring Core, which includes a variety of both conventional and novel assays, will provide uniform assessment of immunologic endpoints. The Clinical Data Management Core provides a vital service of creating dynamic and flexible databases that integrate Projects across multiple Harvard-affiliated hospitals. Finally, a Development Program features five projects, each of which is a candidate for evolution into a full project if promising translational results are obtained. A Career Development Program, which takes a broad view of the development of the physician scientist over the course of a career, is a centerpiece of this application. An administrative structure is in place that will assure oversight, integration, planning, didactic activities, and most importantly, the growth and evolution of the SPORE over the next five years.
Project 1 The overarching goals of this Project are: (1) to utilize our epidemiological understanding of cutaneous melanoma in order to create clinical and population risk estimation and risk stratification; (2) to translate these modeling approaches into patient care in an active clinical setting; (3) to compile genomic profiles of melanoma specimens and correlate these profiles with risk factors for risk stratification. We will explore different risk modeling approaches to better inform primary and secondary prevention strategies. This will lead to stratification for prevention trials, including chemoprevention when agents become available for clinical applications. Extensive epidemiological data from the Nurses' Health Study (121,700 women followed from 1976 to 2000), Health Professionals Follow-up Study (52,000 men followed from 1986 to 2000), and Nurses' Health Study II (116,000 women followed from 1989 to 2001) provide a rich resource on risk factors and the incidence of melanoma in over 230,000 women and 50, 000 men. None of these cohorts has funding to evaluate melanoma risk. Given the unique size and scope of these cohorts, we will examine the ability of risk models to classify individuals with regard to risk of melanoma, and we will evaluate if such a classification of risk can be clinically useful in determining primary prevention strategies and in directing the level of screening surveillance. Based on the above, our specific goals are as follows. We will construct statistical models for melanoma risk, incorporating traditional epidemiological risk factors to stratify population subgroups based on risk. We will construct separate models for women (combining data from the NHS and NHS II cohorts) and men. We will evaluate the models with respect to both goodness of fit (i.e., predicting incidence in subgroups) and discriminatory accuracy at the individual level, and will evaluate the additional discriminatory accuracy gained by including additional risk factors, including genetic markers. We will generate genome wide profile of regional gains and losses in human melanomas as a variable for risk stratification. Genome-wide array CGH (Comparative Genome Hybridization) experiments will be performed on 50 cases of melanoma with no established risk factors, and 50 cases of melanoma from among those with 2 or more risk factors. We will perform these studies at the adult general dermatology clinics affiliated with Harvard Medical School Department of Dermatology (i.e., Massachusetts General Hospital, Brigham and Women's Hospital, Beth Israel Deaconess Hospital).
Project 2 The incidence of malignant melanoma is increasing worldwide at rates that have been reported to be between 3% and 7%. In the United States, the incidence of melanoma is rising faster than any other malignancy. The overall goal of this project is to evaluate whether gene expression profiling can provide additional diagnostic and prognostic information for patients with malignant melanoma. Despite decades of attempts to identify clinical and histological features that would be useful in predicting which lesions were going to metastasize, only limited success can be claimed. The measured thickness (the Breslow measurement) and the presence or absence of ulceration are the strongest predictors of outcome for localized disease. The identification of patients with a high risk of metastasis is important for the surgical management of the patient as well as to determine which patients should be admitted to clinical trials of adjuvant agents as these become available. A comprehensive knowledge of the genes expressed by melanoma may be helpful in management of these patients. The Specific Aims are as follows:
Project 3 Mycosis fungoides is the most common form of cutaneous T cell lymphoma, which is in turn the most common adult non-Hodgkins T cell lymphoma. When the disease is correctly diagnosed in its earliest stages, several treatments are available to arrest disease progression. However, when the diagnosis is made in later stages (e.g., > Stage II), long-term survival is unusual. Current treatments for cutaneous T cell lymphoma (CTCL) are effective primarily when the disease is limited clinically to skin; therapies for disease that clinically involve lymph node, peripheral blood, and other organs are largely palliative There are large deficiencies in our ability to diagnose and treat this non-Hodgkins lymphoma. Moreover, our understanding of the biology of this disease is at best primitive. In the last decade, our understanding of how memory T cells mediate immunosurveillance in different tissues has grown exponentially. Memory T cells that home to skin utilize specific cell surface molecules to exit blood into skin, including CLA, CCR4, and LFA-1. This occurs constitutively, as normal skin contains many such cells, but is greatly facilitated by inflammation, which upregulates the ligands of the above molecules. Our preliminary data and that of others compel us to state the hypothesis that CTCL is a malignancy of the CD4+ skin homing memory T cells. Against this background, we propose the following Specific Aims. To improve our ability to diagnose and treat CTCL, we propose to fully characterize, both functionally and phenotypically, the molecules on mycosis fungoides T cells that are involved with T cell trafficking. We propose that CTCL that involves skin only will express high levels of CCR4 and CCR10 and relatively little CCR7, an L selectin. Conversely, T cells in CTCL that involve lymph nodes will express L selectin, CCR7, and LFA-1, the trinity of homing molecules required for entry into lymph node from blood. Finally, we will test the hypothesis that leukemic CTCL cells do not respond well to skin derived chemokines normally involved in skin homing. In a second Specific Aim, we seek to generate new hypotheses about the biology of CTCL, by using whole genome microarray approaches to human CTCL cells and normal T cells. Taken together, we believe that these studies will improve the accuracy of both diagnosis and prognosis in this disease.
Project 4 Melanoma is a lethal malignancy that is poorly responsive to conventional treatments, but it is potentially an immunoresponsive disease. Several approaches to melanoma immunotherapy have demonstrated the feasibility of eliciting T cell responses to melanoma-associated antigens. In this proposal, we focus on the use of dendritic cells (DC) to induce anti-tumor immunity in patients with advanced melanoma. Because of the manner in which they process protein, express co-stimulatory molecules, and elaborate cytokines that support the development of cytolytic T lymphocyte (CTL) responses, DC are uniquely capable of antigen presentation. They appear to be ideal vehicles for delivery of antigen in tumor vaccination strategies. However, the optimum strategy for delivery has not been determined. Our laboratories have in parallel studied two methodologies for antigen delivery: peptide pulsing of DC with melanoma antigen-derived peptide epitopes, and production of cell fusions between DC and tumor cells. We carried out pre-clinical studies on DC production and function. In doing so, we demonstrated our ability to reliably produce DC in high yields by culturing PBMCs obtained via leukapheresis. Phenotypic and functional analyses showed that these cells express a profile of cell-surface markers characteristic of DC. Concurrently, we utilized DC to verify that specific substitutions of the gp100 280 epitope result in peptides that bind to HLA-A2 with higher affinity than the native epitope. Based on these studies, we are carrying out a phase I, dose escalation trial of peptide-pulsed DC therapy. To date we have shown that this approach is safe. Immunogenicity of vaccination has been documented by the production of CTL specific for the immunizing antigen, and clinical responses have been observed. We also studied the production or DC/tumor fusions. Pre-clinical data demonstrated our ability to generate heterokaryons from DC and breast, ovary, renal cell, and melanoma tumors. Such hybridomas express both DC-derived co-stimulatory molecules and tumor-associated antigens, and reproducibly stimulate the production of anti-tumor T cell immunity in vitro. We are conducting a phase I trial and preliminarily have demonstrated that clinical administration of DC/tumor fusions is safe. DC/fusions also elicit T cell anti-tumor immunity, documented by enhanced interferon secretion in response to stimulation by tumor. Based on these pre-clinical and phase I clinical data, we propose to translate these findings into a phase II, randomized clinical study. Patients will be recruited to both approaches using uniform eligibility criteria at our four institutions. The primary endpoint of the study is the comparison of immunologic data obtained from the two approaches.
Project 5 Cytokine-based therapy has shown considerable promise in melanoma, producing high-quality responses and freedom from relapse in some patients with Stage IV or high-risk Stage III disease. Inteferon ?, FDA approved in 1996, is able to prevent relapse in up to 1/3 of patients with high-risk melanoma, while high-dose IL-2 FDA approval in 1998, produces durable responses in 6% to10% of patients with distant metastases. Efforts to improve upon these approaches have to date been unsuccessful. Perhaps the most promising approach involves combinations of IL-2 and IFN with chemotherapy, so called biochemotherapy. We developed a concurrent biochemotherapy regimen (cisplatin, vinblastine, DTIC, IL-2, and IFN), that produces tumor responses in about 45% to 50% of patients with metastatic melanoma and is sufficiently tolerable and practical to allow for testing in a Cooperative Group setting. This regimen is currently being explored in two large phase III Intergroup trials: a) E3695 that compares the concurrent biochemotherapy regimen with CVD chemotherapy in patients with Stage IV disease; and b) S0008 that compares 3 cycles of biochemotherapy to a year of the standard high dose IFN in patients with Stage III disease. In this project, patients treated on these two trials, as well those treated at DF/HCC institutions on similar treatments, will be asked to provide serial samples of blood, tumor, and lymph node tissue. This material will be analyzed in a variety of ways to determine the mechanisms of response and resistance to cytokine-based and cytotoxic therapy. Evidence of generation of specific immunity directed against melanoma antigens will be assessed using tetramer and ELISPOT analyses against defined melanoma peptide antigens and autologous tumor cells. Results will then be correlated with tumor response, therapy, and disease status. The memory and homing properties of tetramer reactive cells will be explored using multiparameter flow cytometry in order to determine the characteristics of T cells that best correlate with long-acting immunity to melanoma antigens. In addition, T cell receptor signaling properties of peripheral blood T cells will be examined for evidence of tumor based immune dysfunction and the results correlated with treatment, disease status, and effects of therapy. Finally, melanoma tissue will be examined for the presence of various recently discovered factors associated with resistance to apoptosis (STAT 1 and 2, STAT 3, Flip, Apaf-1, etc.) and the findings will be correlated with treatment, treatment results, and disease status. Taken together, this ambitious evaluation should fully define the capabilities and limitations of existing therapies for advanced melanoma and, as such, will pave the way for the application of new treatments, perhaps in combination with the most promising of these standard approaches.
Core 1 The Specific Aims of the Administrative Core are:
Core 2 The purpose of the Biostatistics Core is to provide design and analytic support to SPORE Projects, Developmental Projects, and other Cores. This Core has two specific aims: Specific Aim 1: To provide biostatistical consultation and collaboration for SPORE Projects, Developmental Projects, and Cores The Biostatistical Core will work with SPORE researchers on protocol and experimental design, and will provide estimates of sample size and statistical power. The Core will advise researchers about optimal methods for monitoring ongoing studies. The Core will provide statistical analyses of completed experiments, and participate in manuscript preparation. In addition to these formal collaborative efforts, the Biostatistical Core will be available for ongoing, informal consultation with researchers over the duration of the SPORE. Specific Aim 2: To provide or recommend supporting computational infrastructure. Because an effective informatics infrastructure is critical to the functioning of the SPORE, the Biostatistics Core will work to assure that the storage and accessibility of data used and generated by the SPORE is efficient and effective. The Biostatistics Core will support efficient management of data resources by consulting on database design, helping to design efficient data collection methods and conducting audits of data quality.
Core 3 The purpose of the Tissue and Pathology Core, which will be located within the Departments of Pathology at Brigham and Women's Hospital, Massachusetts General Hospital, and Beth Israel Deaconess Medical Center, is to provide all SPORE investigators with:
Core 4 The Immunologic Monitoring Laboratory (IML) will serve three of the five primary projects and two of the developmental projects of this SPORE application. This laboratory has two responsibilities: 1) It will perform the majority of assays proposed to assess the effects of treatment (e.g., with dendritic cell vaccines, biochemotherapy) on the number, phenotype, and function of melanoma antigen-specific T cells isolated from serial blood specimens, lymph nodes, and tumor biopsies. These assays will consist primarily of peptide- and tumor cell-based ELISPOT analyses, tetramer analyses, as well as assays of cytolytic activity, cytokine induction, and proliferation; and 2) The IML will also be responsible for the studies proposed to evaluate the homing characteristics of cutaneous T cell lymphoma (CTCL) cells (Project 3) and those of lymphocytes obtained from the blood, lymph nodes or tumor tissue of melanoma patients (Projects 4 and 5). These assays consist of extensive multiparameter flow cytometry studies of adhesion molecule and chemokine receptor expression. The laboratory studies conducted by the IML will determine the extent to which various immune-based therapies immunize patients against melanoma antigens and the extent to which the results of surrogate assays of specific immune responses correlate with treatment outcome and other clinical parameters. They will also provide a great deal of new information about the chemokine receptors and adhesion molecules expressed by CTCL cells and by the PBL and TIL of patients undergoing immunotherapy for melanoma.
Core 5 The Clinical Database and Data Management Core (CDDMC) will provide the necessary data collection and management activities for the current and future SPORE projects. The purposes of the CDDMC are:
The CDDMC can provide efficient and consistent quality control and management of the clinical data from the multiple projects over time through a centralized Core facility. Special attention will be paid specifically to information systems, file management, data entry, interaction between the CDDMC and clinical sites, and the preparation of the clinical data for statistical analysis. The CDDMC will manage data on the baseline demographics, clinical characteristics, treatment and outcomes of patients diagnosed with melanoma and cutaneous T cell lymphoma, using the Domain Clintrial v4.3, an ORACLE-based clinical database management system for Projects 2, 3, 4, and 5. Data will be remotely entered at the participating SPORE institutions through a web-interface via Domain Clintrial Connect. The CDDMC will also be interfaced with the Tissue Tracking System for integration of data from the Tissue and Pathology Core and Tissue/Blood Repository. The longitudinal integration of tissue tracking and clinical data will allow correlation of histopathological, genetic, and phenotypical data over time from patients at different stages of melanoma and cutaneous T cell lymphoma.
| ||||||||||||
