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The Wistar Institute
Overall Abstract The intent of the Wistar/Penn SPORE in Skin Cancer is to decrease the morbidity and mortality of skin cancers through improved understanding of the pathogenesis of these diseases using novel, validated molecular biomarkers of risk, progression and prognosis and the development of targeted therapies. This SPORE is focused on major skin cancers: melanoma, cutaneous T cell lymphoma (CTCL) and squamous cell carcinoma (SCC). The overall goals of this SPORE are to address five translationally important areas: 1) Risk assessment. We will test the hypothesis that susceptibility to melanoma stems from complex interactions of multiple factors, including inherited genotypes, environmental exposures, and endogenous pigmentation (Projects 1 and 2). 2) Risk prediction. Genotypes (Projects 1 and 2) will be integrated with existing risk factors to develop prediction models of individual melanoma risk. These models will be important in stratifying individuals for screening and prevention, and to enhance the efficacy of targeted prevention trials. 3) Prognosis. Using data from Projects 1 (alterations in DNA), and 2 and 3 (differences in protein levels) we will develop and externally validate comprehensive, biologically-based prognostic models that will be used in clinical trials and in patient management. 4) Diagnosis. We will develop and validate biomarkers to distinguish melanoma in situ from invasive and/or tumorigenic melanoma (Project 3). 5) Therapy. We will investigate several novel therapies for advanced melanoma and CTCL (Projects 4, 5 and 6). These include: a)targeted therapy for melanoma: molecular targeting of MAP kinase signaling pathways capitalizing on the recent discovery that the majority of human melanomas have an activating mutation in BRAF (Project 6); b)cytokine therapy of CTCL: determination of the mechanisms whereby CTCL becomes refractory to recombinant human IL-12-based therapies and how therapeutic efficacy can be enhanced by cytokines, including IL-2 (Project 5); and c)active and passive immunotherapy of melanoma with lymphocytes and defined tumor antigens: an organotypic model of melanoma will define new cytotoxic T-cell specificities against human melanoma, leading to the identification of novel antigens for vaccine trials and “optimized” adoptive immunotherapy (Project 4). The Wistar/Penn SPORE addresses the most aggressive cancers of the skin. It is focused on melanoma because of its rising incidence and significant potential for lethality. It is the subject of five of the six Projects and three of the five Pilot Studies. CTCL is addressed in one Project and one Pilot Study, and SCC in one Pilot Study. This endeavor represents the synergistic integration of well-established individual research programs towards our collective goals. Project 1: Melanoma has a complex, multifactorial etiology that includes genetic predisposition, UV exposure and somatic genetic changes. This project is focused on the discovery of genetic alterations that play a role in the development of melanoma and the use of those genetic biomarkers in the development of two models. These two models will 1) provide information about risk factors for the probability of having melanoma and 2) risk of metastatic failure in those individuals who do develop melanoma. We will develop the genetic biomarkers for these models by 1) analyzing the association between germline variants in DNA damage response genes and risk for melanoma; and 2) evaluating the mutation spectrum of a panel of receptor tyrosine kinase genes that may play a role in malignant transformation and genome instability in melanomas; and 3) identifying patterns of genomic instability in melanoma using a whole genome approach. We hypothesize that by combining established risk factors with novel genetic biomarkers from these three sources, accurate models for both risk of developing disease and risk of metastatic failure in patients who do develop melanoma can be defined for every individual. These risk profiles also will contain important biological information on melanoma initiation and progression that is needed to develop effective prevention, staging and treatment strategies. The specific aims of this project are: Specific Aim 1: To identify low-penetrance melanoma susceptibility alleles in DNA damage response genes, evaluate their interaction with other genetic variants and risk factors in a case-control study, and develop a multivariate melanoma etiology model. Specific Aim 2: To analyze a panel of 50 primary melanoma cell lines for mutations in candidate receptor tyrosine kinase genes and validate frequent mutation in 50 patient lesions. Specific Aim 3: To develop a novel molecular profile of melanoma using array-based comparative genomic hybridization (aCGH) to characterize amplifications and deletions across the entire genome at 1 Mb resolution. Specific Aim 4: To validate somatic genetic alterations as risk factors for use in prognostic models that will distinguish melanoma patients at minimal and high risk of metastasis, thus requiring different management strategies.
Project 2: The goals of this project are to further develop and validate a series of prognostic models for all melanoma patients presenting with disease apparently confined to the primary site. These models will include both previously validated and new molecular biomarkers and are designed to improve the efficiency of clinical trials (e.g., to establish candidate inclusion criteria and stratification factors appropriate to the outcome of interest) and the quality of patient management (e.g., to protect from over-investigation those with little risk for metastasis). These models will have as their output patient-specific probabilities for the outcome of interest. New models will be developed, tested, and published serially throughout the tenure of the project.
Specific Aim 1: To test innovative biostatistical methodologies and to establish the independent prognostic significance of candidate molecular biomarkers for use in prognostic models to estimate more accurately the likelihood of patient-specific outcomes, we will:
Specific Aim 2; To protect patients with thin ( < 1 mm in thickness) melanomas who are at minimal risk of metastasis from the morbidity and cost of excessive investigation and therapy, as well as to identify those who are at elevated risk and are candidates for trials of more aggressive management, we will:
Specific Aim 3: To better calibrate investigation and management by clinical trialists and clinicians of patients with invasive melanoma of any thickness without clinically apparent regional or distant metastasis (pathological stages IA-IIIC "micro"), we will: 3.1 develop multivariable prognostic models for predicting the likelihood of a sentinel lymph node (SLN) biopsy revealing metastatic disease; 3.2 develop a model that assesses the impact of SLN biopsy (negative, positive, not done) on prognosis; and 3.3 externally validate these models. Specific Aim 4: To optimize surveillance for subsequent primary melanomas, we will:
Project 3: Our long-term objective is to develop new tools for accurate diagnosis of primary melanoma and establish criteria for outcome prediction. Diagnosis of in situ melanoma has remained controversial in recent years due to the lack of molecular tools. It is likely that some in situ melanomas may not progress to invasive melanoma, however there are no molecular tools to distinguish between lesions with and without the potential for progression. Similarly, distinction between invasive and invasive/tumorigenic melanoma by conventional histological criteria remains difficult to conduct by general pathologists and is thus controversial. The development of new molecular tools for diagnosis has been hampered in melanoma despite the availability of new global gene expression technologies. The paucity of new information is due to the small size of most primary melanomas and the necessity for pathologists to have the entire lesion available for histological analyses. The potential risk for metastatic dissemination and disease progression make it also necessary to have the lesions available for several years before material can be processed for experimental studies. To overcome the limitations of available fresh material from primary melanomas for the development of new markers, our laboratories have embarked on two strategies: 1) Develop an experimental model of melanoma in human skin that closely resembles the pathology of patients' lesions. The lesions are derived from human skin grafted to immunodeficient mice and treated by overexpression of three growth factors and concomitant irradiation with ultraviolet (UV) B. This model allows us to work with fresh and frozen tissues for isolation of RNA without further amplification steps. 2) Develop techniques to identify transcripts and proteins from fixed tissue blocks from archival material no longer required for diagnosis or management. In the proposed investigations we will rely for the initial screening and selection studies on the frozen material from experimentally induced melanomas but will perform verification and validation studies with stored paraffin-embedded material from patients. This biphasic experimental design allows us to use state-of-the-art technologies for a unique molecular dissection of primary human melanoma in order to develop new criteria for diagnosis and outcome prediction. In addition, the requirement for UVB irritation in the xenograft model allows us to evaluate the efficacy of various sunscreen strategies in the prevention of melanoma in the human skin xenografts, which we expect will allow us to validate or suggest modifications to strategies for primary prevention of melanoma.
Project 4: Prostate cancer is a complex disease that presumably stratifies into different types of cancers with distinct progression rates, metastatic potentials and prognostic outcomes. We hypothesize that histologically Infiltration of primary melanomas with T lymphocytes is associated with a favorable clinical outcome. This suggests that T cell-based immunotherapies may be beneficial for melanoma patients. However, adoptive cytolytic T lymphocyte (CTL) immunotherapy of melanoma patients and active immunotherapy with CTL-based antigen (Ag)/peptide vaccines have not held promise. These limitations may rest in the selection for immunotherapy of CTL in vitro that do not reflect the Ag specificity of the CTL in situ. CTL traditionally are selected in two-dimensional mixed lymphocyte-tumor culture (MLTC) directly on plastic surfaces. We have developed the three-dimensional human melanoma organotypic culture model (melanoma reconstruct) in which CTL are cultured under tissue-like condition. Our preliminary studies suggest that the T cell receptor (TCR) repertoire in tumor reconstructs more closely resembles the repertoire of the T cells in situ as compared to the TCR repertoire in MLTC. However, the two culture systems are complementary. Thus, the chances of generating T cells with in situ (tissue)-like TCR are enhanced when T cells from both culture systems are combined, compared to either culture alone. Our major goal is to develop, including the melanoma reconstruct as a culture system, improved CTL-based adoptive and active immunotherapies against melanoma. Specifically we will: 1. Compare the phenotypic (CD markers, TCR, adhesion and apoptosis-related molecules, HLA, chemokines, chemokine receptors) and functional characteristics (proliferative and lytic activity; cytokine production) of CTL derived from fresh tissues in the reconstruct or MLTC. 2. Clone Ag(s) recognized by CTL that are highly cytolytic and express the original TCR found in situ. These Ags have vaccine potential for melanoma patients and may boost pre-existing immune responses. 3. Identify chemokines and chemokine receptors that are involved in CTL migration toward tumor cells in the reconstruct. This involves: i) Selection of migrating CTL and ii) blocking of chemokines and their receptors to determine their role in CTL migration. Chemokines relevant in CTL migration in the reconstruct may be used to enhance anti-tumor effects of adoptive and active immunotherapy. The proposed studies open new approaches to CTL-based adoptive and active immunotherapies, which may be initiated during the third year of the SPORE. Project 5: The objective of this proposal is to evaluate novel approaches for the treatment of melanoma through the molecular targeting of essential signaling pathways. To accomplish this translational objective, we have performed preclinical studies to identify and validate potential candidate targets in cellular proliferative and survival pathways, and based on these results we propose to conduct a series of clinical trials to determine the efficacy and safety of inhibiting these pathways in patients with advanced melanoma. Overall, the goal of the first 2 specific aims is to capitalize on the recent discovery that the majority of human melanomas are associated with an activating mutation in BRAF and to select promising new agents which target the Raf pathway. For the first clinical trial, we have selected BAY 43-9006, a small molecule inhibitor of the Raf family of protein kinases to evaluate in a phase II study in patients with metastatic melanoma. In specific aim 2, correlative laboratory studies will investigate the ability of BAY 43-9006 to inhibit its specific molecular target, Raf kinase, and we will assess whether the degree of inhibition of the RAF/MEK/ERK cascade in tumor tissue and surrogate tissues correlates with clinical response. Further studies will determine the relationship between clinical response to BAY 43-9006 and the mutational status of BRAF in the tumor samples and pharmacokinetic and pharmacodynamic analyses of BAY 43-9006 will be performed to determine kinetic characteristics that are effective in target inhibition. In specific aim 3, we will further investigate the role of survival pathways in melanoma by conducting preclinical and clinical studies to determine whether the PI3-Kinase/Akt/TOR survival pathway represents an additional site for therapeutic intervention in melanoma. While the primary objective of this proposal is therapeutic, that is, identifying safe and beneficial treatments for patients with melanoma, we anticipate accumulating further insights into understanding the basic biology of melanoma and signaling pathways as a byproduct of these preclinical and clinical studies.
Core A: The Administrative Core is designed to provide overall leadership for the SPORE. The PI and Co-PI, with the support of an administrative secretary, organizes and manages the different components of the program. This includes frequent interactions with the Co-PI, Project Leaders, Co-Investigators from the Projects and Pilot Studies, and Core Leaders, internal program review and planning, external review, scientific seminar series, and overall representation of the needs of the scientific and administrative programs. Core A communicates with the external and internal advisors, the Cancer Center Directors of both the Wistar Institute and the University of Pennsylvania, and the NCI program staff. This Core serves the needs of the entire program on the SPORE in Skin Cancer for the institutions on the Wistar/Penn campus and the general scientific community.
Core B: Principal Investigator(s): David E. Elder, M.B., Ch.B., F.R.P.A. The Tissue and Pathology Core will collect tissue samples from human skin tumor specimens, for use in the SPORE projects and pilots, and for the use of other qualified investigators as determined by the Tissue and Resource Allocation Committee in accordance with the SPORE guidelines. Procedures have been developed for identifying and obtaining informed consent from patients to collect tissue at the time of surgery, as well as for procurement, processing, storage, quality control, gross and microscopic pathological evaluation, and allocation of samples that will ensure optimal utilization and distribution of the limited tissue samples. In addition, the Core will provide expertise to the Projects for the development of in situ imaging and microdissection techniques in sections of human skin tumors for use in the projects, and for the development of techniques for expert pathological interpretation of histological data, using immunohistochemical methods with a variety of antigen retrieval techniques, as well as in situ hybridization using oligonucleotide probes, laser cutting microdissection, and nucleic acid amplification techniques.
Core C:
Biometrics The goal of the Biometrics Core is to provide SPORE investigators access biostatisticians who have experience with biostatistical methodology and their application to research studies in cancer of the skin. The Biometrics Core staff will provide expertise in research methodologies necessary to design and implement rigorous research studies in Specific Aim 1. Through Specific Aim 2 they will provide expertise in informatics necessary to support efficient database development and database linkage, as well as expertise in statistical programming necessary to implement sample designs and both descriptive and inferential statistical analyses for SPORE studies. By way of Specific Aim 3 they will provide expertise in statistical methodology critical in the evaluation of research hypotheses and in the development of statistical models specified by the research objectives of the SPORE studies. Lastly, through Specific Aim 4, they will provide oversight, maintenance and quality assurance for data stored in the SPORE Database Library, facilitating access to it for SPORE-related inquiries and uses. Through these specific aims the Biometrics Core will insure that SPORE-related studies will have high quality study designs and statistical analysis plans that will provide a solid foundation for statistical inferences. Recruitment of fellows and staff will occur through the planned educational program and by systematic recruitment activities. These individuals will be monitored by a specific Career Development committee, members of the SPORE, and by over 33 non-SPORE senior investigators whose research interest and accomplishments reflect prostate cancer research concerns. In addition to SPORE grant support, this program will be supported by extensive institutional resources. The Wistar Institute Meenhard Herlyn, D.V. M., D.Sci. David E. Elder, MB, Ch.B., F.R.P.A. DuPont Guerry, M.D. Klara Balint, M.D. Brian Czerniecki, M.D., Ph.D. Rosalie Elenitsas, M.D. Arupa Ganguly, Ph.D. Phyllis A. Gimotty, Ph.D. Dorothee Herlyn, D.V.M. Peter Kanetsky, Ph.D. Michael Ming, M.D. Peter O’Dwyer, M.D. Timothy R. Rebbeck, Ph.D. Lynn M. Schuchter, M.D. Leslie Shaw, Ph.D. Rajasekharan Somasundaram, Ph.D. Rolf Swoboda, Ph.D. David Tuveson, M.D. Barbara L. Weber, M.D. Xiaowei Xu, M.D., Ph.D. Tianqian Zhang, Ph.D. |
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