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THE WISTAR INSTITUTE
Overall Abstract
Principal Investigator(s): Meenhard Herlyn, D.V.M.
Cancers of the Skin investigated in this SPORE include melanoma, cutaneous T cell lymphoma, and squamous cell carcinoma (SCC) and basal cell carcinomas (BCC). The projects and pilot studies are based on a broad existing infrastructure of active basic and translational research in cancers of the skin. They span the fields of cancer epidemiology, biology, immunology, pathology, and therapy to assess cancer risk, diagnosis and detection, prognosis, treatment, and treatment outcome. Immune surveillance genes are investigated as candidate melanoma susceptibility genes (Project 1). Prognostic models are developed for primary melanoma to better design patients' treatment and follow-up (Project 2). Lymphocyte populations from melanoma patients are analyzed to develop new strategies for passive and active immune therapy (Project 3). Patients with cutaneous T cell lymphoma are evaluated that fail therapy with the cytokine IL-12 (Project 4). A melanoma vaccine is being developed that utilizes new principles in antigen presentation (Project 5). Developmental funds will be made available to increase the number of investigators in translational studies. Pilot studies are designed for melanoma, SCC and BCC for disease diagnosis, detection and therapy. All pilot studies utilize novel technologies and approaches to extend the existing infrastructure allowing the group to develop new projects in cancer prevention, diagnosis and therapy. Resource centers provide the investigators with support for all projects. By building on a strong existing infrastructure, which will be further strengthened through this SPORE, the proposed studies are expected to have a major impact on the control of Cancers of the Skin.
Project 1
Candidate Susceptibility Genes for Melanoma: Immune Surveillance Genes
Principal Investigator(s): Meenhard Herlyn, D.V.M.
Co-Investigator(s): Timothy Rebbeck, Ph.D.; Anne-Marie Martin, Ph.D.;
Peter Kanetsky, Ph.D.
Predisposition to develop melanoma results from a complex, multifactorial etiology that involves genetic factors, endogenous physiology, and exogenous exposures. The interaction of these factors to cause melanoma may differ by age, race, or family history. Knowledge about multiple risk factors in melanoma etiology will aid in the identification of individuals at increased risk for developing melanoma. Ultimately, this knowledge may facilitate melanoma prevention strategies. We propose to study candidate genetic biomarkers of melanoma risk, and determine their association with melanoma phenotypes.
The underlying biological hypothesis of the proposed research is that susceptibility to develop melanoma stems from the complex interactions of multiple factors, including inherited genotypes associated with immune surveillance. To address this hypothesis, we will use a multidisciplinary approach that involves methods drawn from molecular biology and epidemiology to evaluate the role of genes involved in melanogenesis and development of a melanoma. We propose to consider the role candidate melanogenesis genes (i.e., cytokines and other immune surveillance genes (ISG)), the interaction of these genes and other melanoma risk factors (e.g., skin/hair/eye pigmentation, previous UV exposure, history of dysplastic nevi, and family history of melanoma) in the etiology of melanoma. Using the existing infrastructure of the Pigmented Lesion Group at the University of Pennsylvania, we propose a case-control study that will directly address the complex, multifactorial etiology of melanoma that involves the interaction of genotypes involved in immune surveillance. This study will address a number of specific hypotheses. First, we will characterize the frequency distribution of alleles at candidate susceptibility genotypes. Second, we will evaluate whether genotypes are associated with tumor characteristics including stage, grade, and age at diagnosis. Finally, we will evaluate whether these genotypes are involved in melanoma etiology, and whether these genotypes interact with one another and other melanoma risk factors.
In order to address these hypotheses, we will undertake a study using the extensive resources of the Pigmented Lesion Group at the University of Pennsylvania. The sample will consist of 1000 melanoma cases and 1000 controls. Risk factor information will be obtained by questionnaire, a DNA biosample will be collected using a non-invasive cheek swab method, and diagnostic pathology information will be collected using a systematic approach. Analyses will be undertaken to evaluate the role of candidate genotypes and other risk factors in melanoma etiology, including genotype by environment interactions.
Project 2
Clinically Useful Prognostic Models in Primary Melanoma
Principal Investigator(s): Meenhard Herlyn, D.V.M.
Co-Investigator(s): Phyllis Gimotty, Ph.D; DuPont Guerry, M.D.; Michael Ming, M.D.
Staging and therapy of cutaneous melanoma patients with sentinel lymph node (SLN) biopsies and the use of systemic adjuvant therapy in those with high risk primaries and/or regional lymph node metastases lately have become commonplace, although neither has been shown unequivocally to improve survival. It is both timely and important to develop prognostic models that have potential utility for the design of efficient clinical trials and for clinical management of patients with melanoma. The objective of this project is to develop prognostic models using a rigorously documented and carefully followed cohort of over 5,500 melanoma patients assembled since 1972 and an archive of tissue blocks containing a representative sample of their primary lesions. The application of novel biostatistical methods using clinical and new immunohistologic data will allow us to achieve our explicit goals. To protect patients with "minimal risk" melanomas from the morbidity and cost of excessive investigation and therapy, in the first specific aim we will test the hypothesis that invasive melanoma without metastatic capacity can be identified using currently available, easily employed, and readily interpreted clinical, histologic and immunohistologic prognostic variables. To better calibrate investigation and management of patients with metastatic capacity by clinical trialists and clinicians, in the second specific aim we will develop multivariable prognostic models for predicting the likelihood of a SLN biopsy revealing melanoma and of metastasis–free survival after regional surgical staging and therapy. To optimize surveillance for additional primary melanomas in follow-up, in the third specific aim we will develop multivariable prognostic models for predicting the occurrence of a second primary. To promote the use of these models by clinical trialists and clinicians, we will develop individualized patient probabilities of the occurrence of clinically relevant events that can be used in trial design and clinical decision making.
Project 3
T Cell-Based Immunotherapy in an Organotypic Melanoma Culture System With Clinical Relevance
Principal Investigator(s): Meenhard Herlyn, D.V.M.
Co-Investigator(s): Dorothee Herlyn, D.V.M.; Jian Li, Ph.D.; R. Somasundaram, Ph.D.; Rolf Swoboda, Ph.D.; Tianquian Zhang, Ph.D.; Brian Czerniecki, M.D.
Critical issues in cancer immunotherapy are readily apparent in melanoma and include the findings that: 1) melanomas grow in patients in spite of being infiltrated with lymphocytes; ii) lymphocytes that lyse melanoma targets in vitro often do not demonstrate therapeutic effects after adoptive transfer to patients in vivo; and iii) induction of cytolytic T lymphocytes (CTL) in melanoma patients with CTL-defined antigens (Ag)/peptides did not correlate with clinical responses. Understanding the mechanisms underlying these phenomena holds the promise of revolutionizing CTL-based immunotherapies. However, these mechanisms can not be addressed directly in vivo in patients and thus far relevant in vitro models have been lacking. In the proposed studies our major goal is to use the human melanoma organotypic (dermis reconstruct) culture model to develop novel CTL-based adoptive and active immunotherapies against melanoma. Our preliminary studies have demonstrated that CTL which were generated against melanoma cells in a mixed lymphocyte/tumor cell culture (MLTC) are able to lyse the melanoma cells in the human melanoma/dermis reconstruct at a reduced effector-to-target (E:T) cell ratio. Our Specific Aims are to: 1) Determine in vitro in the reconstruct model the factors that play a role in the activation or inhibition of CTL activity against melanoma cells in vivo in patients. Identification of these factors will reveal new ways to make effective adoptive immunotherapy of cancer patients. 2) Evaluate the potential of the human dermis reconstruct model for the generation of new CTL from peripheral blood mononuclear cells (PBMC) or tumor-infiltrating lymphocytes using cultured or fresh melanoma cells as stimulants and compare the CTL activity generated in this model with that generated in MLTC. These studies are likely to isolate the "optimal" CTL which has been induced (committed) under in vivo-like conditions eliminating the artifacts of in vitro culture systems. 3) Clone selected Ag recognized by the CTL that are highly effective in the reconstruct model with emphasis on the CTL induced against fresh melanoma specimens. The Ag recognized by optimally effective CTL identified in the reconstruct will be cloned using the well established COS cell system. 4) Design and implement clinical trials with adoptively transferred CTL and CTL-defined Ag in melanoma patients. The proposed studies are translational, based on the development of a novel human organotypic culture, offering novel immunotherapies of melanomas using adoptive transfer of CTL or CTL-defined vaccines.
Project 4
Interleukin-12 Defects and In Vivo Effects of Interleukin-12
Principal Investigator(s): Meenhard Herlyn, D.V.M.
Co-Investigator(s): Alain Rook, M.D.; Maria Wysocka, Ph.D.; Mariusz Wasik, M.D.
Cutaneous T-cell lymphoma (CTCL) is a clonally-derived, skin-invasive malignancy of CD4+ T-lymphocytes with the phenotype of mature helper T-cells. Patients with advanced forms of CTCL characterized by multiple tumors or peripheral blood involvement typically have a poor prognosis. Our previous work has demonstrated that advanced CTCL is characterized by prominent immunologic defects including depressed cell-mediated immunity. A marked defect in IL-12 production in CTCL has also been noted, which may play a role in depressed cell-mediated immunity. Because evidence exists for an antitumor T-cell response and since IL-12 is pivotal in stimulating cytotoxic T-cells, several clinical trials have been performed with recombinant IL-12 with 10 of 23 evaluable patients responding. Key observations in these small studies were 1)responses were rapid but were often eventually associated with an IL-12 refractory state 2) the latter may be associated with downmodulation of IL-12 receptors on lymphocytes 3) regressing lesions were intensely infiltrated with cytotoxic T-cells and 4) more robust responses occurred at the lower dose of 100 ng/kg used in the phase I trial. This proposal plans to study the in vivo effects of IL-12 in another multicenter trial which will combine IL-12 with escalating doses of IL-2. We propose to investigate the in vivo mechanisms of action of IL-12 by studying 1) skin infiltrating immune cells, 2) cytokine production in situ in skin lesions, and 3) extent of apoptosis within active skin lesions prior to and during IL-12 therapy and correlate this with lesion regression. We will also examine IL-12 responsive NK cell activity and interferon gamma production in concert with IL-12 receptor expression and IL-12 signal transduction prior to and prospectively during therapy to determine if downmodulation of receptors or alteration of Jak-Stat pathways accounts for tolerance to the clinical effects of IL-12. We also propose to further investigate the defect in CD40 ligand expression on malignant T-cells as an underlying mechanism for IL-12 production defects and to use recombinant CD40 ligand as an additional adjuvant to potentially induce synergistic antitumor immune responses.
Project 5
Novel Melanoma Vaccine for Patients with Metastatic Melanoma
Principal Investigator(s): Mark Tykocinsky, M.D.
Co-Investigator(s): Aoshuang Chen, Ph.D.; Carl June, M.D.; Lynn Schuchter, M.D.
This proposal seeks to clinically evaluate a novel tumor cell vaccination strategy, with human melanoma as the preferred tumor target. The "costimulator-expressing" tumor cell vaccine advocated here breaks new ground in a number of ways, by virtue of its: 1) reliance on protein transfer, as opposed to gene transfer, for neo-expressing costimulators on the tumor vaccine cells, which enables unprecedented quantitative control of surface costimulator levels and simplifies the simultaneous delivery of more than one costimulator; 2) combinatorial use of costimulators directed towards the activation of multiple anti-tumor immune effectors, including T, NK, and dendritic cells; and 3) shift to intratumoral administration of the multi-component costimulator therapeutic, which constitutes a move to in vivo, from the more typical (and inherently more cumbersome) ex vivo, costimulator engineering of tumor cells. The preliminary data document the feasibility of applying an innovative Fc fusion protein transfer method for this "in situ" cancer vaccination in a murine tumor model. The major observation is that the injection of a "tetra-costimulator" combination (with costimulators chosen to activate T, NK, and dendritic cells), in the form of palmitated protein A:costimulator·Fc conjugates, directly into a tumor bed results in both local tumor regression (with a prolific local inflammatory response) and signs of systemic anti-tumor immunity. The four specific aims of this proposal now seek to develop this vaccination strategy in greater depth and move it into clinical trials. The first two preclinical aims call for casting a wider net amidst the pool of available costimulators and identifying ones that are optimal for melanoma (Specific Aim #1), along with resolving dosing and other issues that are peculiar to the intratumoral costimulator inoculation method (Specific Aim #2). In turn, the baseline information gained will enable a Phase I clinical trial, which encompasses the evaluation of toxicity (under Specific Aim #3) and immunological responses (under Specific Aim #4) in patients with metastatic melanoma, whose tumors are directly injected with the preferred "multi-costimulator paints."
Project 6
Gene Expression Profiling of Experimentally Induced Human Melanocytic Lesions and Correlation with Precursor and Primary Melanoma Lesions From Patients
Principal Investigator(s): Meenhard Herlyn, D.V.M.
Co-Investigator(s): Carola Berking, M.D.; Pat VanBelle; David Elder
The etiological role of ultraviolet (UV) light in the development of melanocytic lesions has been established through epidemiological and experimental studies. However, specific molecular and biological effects of UV light on skin that lead to melanocyte transformation are not known and have been difficult to study in the human system due to lack of appropriate models. Our laboratory has recently established an experimental model of melanocyte transformation that allows systematic profiling of UV light-induced aberrations in human skin. Full thickness human skin is grafted to severe combined immunodeficiency mice and exposed to a combined treatment of bFGF and UV light over a period of 1-3 months. Intradermal injections of adenoviral vectors for bFGF once weekly ensure a stable expression of bFGF protein in the skin. UVB irradiations of the human skin xenografts were performed at a dose of 50 and 250 mJ/cm2 three times weekly. Hyperplastic and atypical melanocyte lesions developed after 2-3 months of treatment. In this study, global gene expression in the experimentally transformed melanocytes will be analyzed by cDNA array technology after amplification of total RNA from microdissected cells. Likewise, melanocytic precursor (nevi and dysplastic nevi) and primary melanoma lesions from sun-exposed and sun-protected body areas from patients with and without a history of intense sun exposure will be subject to gene expression analysis. Comparison between experimentally induced and clinical lesions by correlation studies and cluster analysis will detect melanocytic gene expression changes associated with UV light exposure and indicative of transformation towards melanoma development. We expect from these studies the identification of UV light associated markers of melanocyte transformation that can be of diagnostic and prognostic relevance.
Core A
Administration
Principal Investigator(s): Meenhard Herlyn
The Administrative Core is designed to provide overall leadership for the SPORE. The 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, 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. This Core serves the needs of the entire program on Cancers of the Skin for the institutions on the Wistar/Penn campus and the general scientific community.
Core B
Tissue and Patient Accural
Principal Investigator(s): Meenhard Herlyn, D.V.M.
Co-Investigator(s): David Elder, Rosalie Elenitsas, and Michael Ming
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 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, in situ hybridization using oligonucleotide probes, laser cutting microdissection, and nucleic acid amplification techniques.
Core C
Biometrics
Principal Investigator(s): Phyllis Gimotty, Ph.D.
Co-Investigator(s): Aiyi Liu, Ph.D.
The goal of the Biometrics Core is to provide SPORE investigators access to a team of 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. In 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. In 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, in Specific Aim 4, they will provide oversight, maintenance and quality assurance for 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.