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Skin Cancer SPOREJohn Kirkwood, MD, Principal Investigator The University of Pittsburgh Cancer Institute is conducting a Specialized Program of Research Excellence (SPORE) in Skin Cancer. The overall goals of the SPORE are to improve the detection and treatment of skin cancer. The SPORE includes five translational research projects in skin cancer, four cores including an administrative core, a developmental research program, and a career development program. The Skin Cancer SPORE uses an interdisciplinary approach to meet its objectives by carrying out projects with co- investigators in basic, applied, and clinical science. It is also organ-specific; all translational research projects test hypotheses about skin cancer biology, susceptibility, detection, or treatment with a long-term goal of improving treatment outcomes for patients diagnosed with skin cancer. The main projects are:
The research cores are the Immunological Monitoring and Cellular Products Laboratory/Tissue Microarray (IMCPL/TMA) Core, the Biostatistics Core and the Informatics Core. These research cores will support the main research projects, developmental research projects, and the career development investigators in carrying out translational skin cancer research. The Administrative Core will solicit feedback from the Internal and External Scientific Advisory Boards and provide scientific, regulatory, and fiscal oversight for the SPORE program. The Skin SPORE investigators will work together to synergistically achieve the goals of the program and will also interact with investigators from SPOREs at other institutions to improve the outcome of patients with skin cancer. Contact:
Project 1 Project 1 is a corollary biomarker evaluation we propose as a study nested in the ongoing ECOG 1697 intergroup trial. E1697 evaluates adjuvant high dose IFN-α2b given for 4 weeks IV 5/7 days a week vs. observation in patients with resected melanoma of intermediate risk: (1) T2b N0, (2) T3a-b N0, (3) T4a-b N0, (4) T1-4 N1-2a, (microscopic). Recent evidence suggests an association between autoimmunity and favorable melanoma outcome after high-dose IFN-α2b. We propose to first evaluate the presence of autoantibodies to multiple autoantigens of normal tissue as well as melanoma, utilizing ELISA or Luminex analysis of serial blood samples from 300 patients at baseline, 1, 3, 6, 9 and 12 months on IFN or observation, in E1697. This evaluation of autoimmunity as a marker of outcome of adjuvant IFN-α2b will determine the predictive value of autoantibody responses individually and collectively. Patients may be genetically predisposed to developing autoimmunity with IFN-α2b therapy, and evidence supports specific HLA genotypes and polymorphic variations in the CTLA-4 and FOXP3 genes as potential immunogenetic markers that may predict the capacity for autoimmunity, and therefore, benefit from therapy with IFN-α2b. We will therefore determine whether patients are predisposed to autoimmunity induced by IFN-α2b based on these immunogenetic markers in PBMC using PCR-based assays, and determine whether a genetic signature of adequately high sensitivity and specificity may predict development of autoimmunity induced by IFN-α2b treatment. We hypothesize that the analysis of the immune responses of patients who have developed autoimmunity, in the context of their MHC class I/II composition, will allow the definition of new and more relevant antigens for future vaccine immunotherapy. In the context of the proposed studies we will measure current candidate serum markers including S-100, LDH, CRP and MIA levels by ELISA, as well as a panel of cytokines by multiplex analysis for comparison to the autoimmune panel we have focused upon in this project. This study will evaluate immunologic, genetic, and other biomarkers to classify melanoma patients into groups with different prognosis and therapeutic potential. We will attempt to develop prediction models to identify groups of patients that are more or less likely to benefit from adjuvant therapy.
Project 2 This application will build on progress made in our previous tumor antigen targeting trials to test a new genetically engineered dendritic cell-based vaccine regimen designed to more potently activate CD8 and CD4 T cells specific to multiple melanoma antigens. We will couple this vaccine trial with thorough immunological monitoring to study T cell responses to the vaccine and the importance of determinant spreading for clinical response. We hypothesize that vaccination with multiple full length tumor antigens will activate a broad range of CD4 and CD8 T cells, and that in the subset of patients who further activate and diversify their T cell response to include other antigens expressed by their tumor (or undergo “determinant spreading”), objective clinical response will be observed. We also hypothesize that systemic IFNa delivered after the vaccine will boost the vaccine-specific T cell responses. Specific Aims: Specific Aim 1. Conduct an Antigen-Engineered DC Trial with an IFNa Boost. We will treat 30 subjects with antigen engineered DC and randomize half to receive an IFNa boost. Specific Aim 2. Assess the Biology of the CD8 and CD4 Immune Responses to Immunizing Antigens MART-1, Tyrosinase and MAGE-A6. We will follow CD8 and CD4 T cell responses to the three immunizing antigens, as well as the adenovirus vector. We will also investigate TIL responses to the immunizing antigens and tumor antigen expression in accessible tumor deposits. Specific Aim 3. Assess Determinant Spreading. We will also follow the CD8 and CD4 T cell responses to defined melanoma-associated antigens not included in the vaccine but commonly expressed by tumors (including gp100) and uncharacterized antigens expressed by autologous tumor to determine the importance of determinant spreading to objective clinical response.
Project 3 Type-1 polarized DCs (aDC1s) show selectively enhanced ability to induce functional Th1 and CTL (Teff) cells, when compared to the current “standard” of clinically-used DCs (sDC). We are currently implementing a phase I/II clinical trial evaluating the relative abilities of αDC1 and sDCs to induce melanoma-specific immunity in stage III/IV melanoma patients (UPCI 03-118; funded by an independent R21 grant to Kalinski/Kirkwood). Our new preliminary data demonstrate that aDC1 and sDCs induce different sets of T cell-associated chemokine receptors (CKRs), with aDC1s being superior in inducing CXCR3 andCCR5, the CKRs implicated in tumor-entry of melanoma-specific T cells. We propose to analyze the mechanism of the differential ability of aDC1 and sDCs to induce distinct CRK expression in tumor-specific T cells, as the fourth DC-related signal essential for the efficacy of DC-based cancer vaccines. We will test the hypothesis that DCs maturing in different environments, in addition to their differential ability to induce type-1 versus type 2 responses (delivery of signal 3), can also differentially regulate the expression of CKRs in tumor-specific immune cells (delivery of signal 4). We will pursue three Specific Aims: Specific Aim 1. Determine the mechanism of DC-dependent regulation of T cell chemokine responsiveness in vitro. We will test the hypothesis that, in contrast to the current paradigm, the induction of a distinct CKR profile is regulated in a different mechanism than the Th1/Th2 commitment. Specific Aim 2. Demonstrate that DCs maturing in different conditions (aDC1 v. sDC) differentially regulate chemokine responsiveness of melanoma-specific T cells in vitro and in vivo. We will directly test the hypothesis that aDC1 and sDCs can (differentially) modulate the CKR patterns in melanoma-specific T cells, and can revert the established CKR patterns on the tumor-induced T cells in melanoma patients. Specific Aim 3. Demonstrate the differences in local chemokine production and the character of local T cell infiltrates between DTH sites, primary, and metastatic tumor lesions. We will test the hypothesis that the tumor tissues constitute a biased chemokine environment and the hypothesis that primary and metastatic tumors represent different chemokine environments, contributing to often ineffective immune control of tumor metastases, even in DTH-positive patients.
Project 4 We have recently determined that more than 90% of melanomas overexpress the receptor tyrosine kinase (RTK) EphA2, with the EphA2 level of overexpression associated with metastic phenotype and an adverse clinical prognosis. Cross-linking of tumor cell-expressed EphA2 molecules using either ligand Ephrin A1-Ig fusion proteins or activating anti-EphA2 antibodies results in RTK phosphorylation, followed by receptor internalization, c-CbI-dependent ubiquitination and proteasome-dependent degradation. As a consequence, agonist-treated tumor cells acquire a more benign phenotype, and based on our preliminary data, they conditionally up-regulate their expression of EphA2-derived epitopes presented in MHC class I complexes. Using Eph-A2-specific CTL lines and clones, we have shown in preliminary data that treatment with these EphA2 ligand agonists results in improved recognition and killing of tumor cells by anti-EphA2 CD8+ T cells in vitro and in vivo in Hu-SCID models. We have also recently observed that pharmacologic inhibition of protein tyrosine phosphatases (PTP) or HSP90 function also serves to increase EPhA2 proteasomal processing, theoretically making Eph-A2 peptides accessible to the MHC class I biosynthetic pathway. We hypothesize that EphA2 ligand agonists and PTP/HSP90 inhibitors may act synergistically in promoting enhanced tumor cell recognition by CTLs. As a consequence, we hypothesize that combinational immunotherapies consisting of 1) EphA2-based vaccines designed to elicit specific CTLs and 2) the conditional activation of EphA2 degradation and proteasomal processing via locoregional administration of EphA2 ligand agonists or PTP/HSP90 inhibitors will result in improved anti-melanoma efficacy. Our Specific Aims are to: Evaluate the ability of “agonists” that promote EphA2 proteasomal processing to sensitize melanoma cells to anti-EphA2 CD8+ T cell recognition in vitro (AIM 1); Test the hypothesis that combinational immunotherapies targeting the conditional proteasomal processing of EphA2 are safe and more effective than single modality therapies in mouse models in vivo (AIM 2); and Design and perform a phase I clinical trial of a combinational therapy involving DC1/EphA2 peptide immunization and conditional augmentation of tumor presentation of EphA2-derived epitopes in HLA-A2+ patients with advanced-stage melanoma (AIM 3).
Project 5 Numerous pre-clinical and clinical studies, including our own, demonstrate that DC-based tumor immunotherapies can induce potent tumor specific immunologic and clinical responses. However, complete clinical responses have been rare, and the improvement of DC-based immunotherapies is the focus of considerable effort. Increasing evidence suggests that tumor induced immunosupression, and the presence or induction of regulatory T-cells (Tregs), may limit the efficacy of anti-tumor immunization strategies. Our hypothesis is that Tregs play a critical role in limiting DC-based immunization against tumors, and that the reduction/removal of Tregs prior to immunotherapy may result in enhanced anti-tumor immunity and improved therapeutic efficacy. The studies we propose here will provide the experimental foundation for this approach and enable a direct test of this hypothesis through clinical trials designed to evaluate combined immunization/Treg depletion approaches for this immunotherapy of patients with advanced state cutaneous T-cell lypmphoma (CTCL). In our preliminary studies, we have developed and evaluated a DC-based immunization strategy for the immunotherapy of patients with CTCL. To construct an autologous tumor vaccine, we have utilized patient-derived matured and “polarized” DCs loaded with autologous circulating tumor cells from leukemic CTCL patients. Our preliminary data demonstrates that immunization of a patient with end-stage Sezary Syndrome SzS resulted in significant anti-tumor immune response and a complete clinical response. The studies we propose here are designed to continue the logical progression of the development of this immunization strategy by identifying and obviating immunosuppressive mechanisms in the host that are likely to limit vaccine efficacy. Our preliminary data demonstrate that CTCL patients have elevated levels of Tregs compared to normal controls. In this project we will evaluate and characterize Treg populations in CTCL patients, and will develop strategies to reduce or eliminate Tregs prior to immunization using already approved chemotherapeutic agents. These studies will provide the basis for the in vivo evaluation of the effectiveness of combined immunization and Treg depletion therapies for CTCL, and future analogous approaches for the treatment of human cancers.
Core A: Administrative Core The Administrative CORE A will oversee all administrative and scientific activities of the UPCI Skin Cancer SPORE. Its responsibilities include the coordination of the internal scientific and clinical conduct and all financial, regulatory, and informatics components of the research that is planned and in progress. CORE A will be responsible for internal and external communications regarding the weekly clinical-pathology conferences of the SPORE investigators, and alternate-weekly clinical-scientific reviews of all protocols of investigation for the SPORE, as well as the monthly data safety reviews. The Administrative CORE will oversee the clinical coordination, data management, and research tumor registry database that serves as annotation of all tissue bank specimens of the protocol-driven Immunological Monitoring and Cellular Products Laboratory /Tissue Microarray (IMCPL/TMA) (CORE B) of this SPORE. The Administrative CORE will be responsible for all communications with the external and internal Scientific Advisory Boards, and NCI personnel. It will submit all reports upon SPORE activities, and coordinate an annual retreat for the SPORE investigators, as well as the travel of SPORE investigators to the annual NCI-sponsored SPORE meeting. The Administrative CORE will fund travel of selected investigators to present their research results at scientific conferences, and assist investigators in the preparation and submission of manuscripts for publication, maintaining the informatics associated with this SPORE, including an annotated database expanded out of the tumor registry and linked to the protocol-based and CORE B Tissue Bank resource, that include more than 7000 patients with melanoma and several thousand tissue specimens collected upon protocols of investigation over the past 20 years. CORE A will maintain a computerized library of pertinent references of the investigators, and will coordinate activities of the SPORE in relation to the UPCI and with the relevant Departmental Chairs responsible for this Interdisciplinary Program to facilitate the research productivity and complementarities, and synergy within the institution an in relation to the other skin SPOREs and the UPCI, and Cooperative Groups. The Administrative CORE will interact with the Patient Representatives, and communicate with the NCI Program Office, to ensure that the SPORE guidelines are adhered to and that the mandate for translational research is accomplished efficiently and in a timely manner.
Core B: Immunologic Monitoring and Cellular Products Laboratory/Tissue Microarray Core (IMCPL/TMA) This laboratory is a specialized facility at the University of Pittsburgh Cancer Institute (UPCI), which is dedicated to the state-of-the-art evaluation of immune responses prior to, during and after therapeutic interventions in patients with cancer. In addition to generating cellular products for human therapy, it also provides specialized morphology services. In its role as Core B for the SKIN SPORE, the IMCPL will assume responsibility for supporting the biotherapy-based clinical trials proposed by the projects. Core B, functioning as a GMP facility, will culture and characterize dendritic cells (DC) for patient therapy and prepare vaccines by pulsing these DC with peptides or proteins or infecting DC with AdV, as specified in the clinical protocols associated with the SPORE grant. Core B will be responsible for quality and sterility of the DC-based vaccines. It will also procure and process all body fluids and tissues harvested in the course of the clinical trials. Tissue specimens will be used for microarray preparation and immunohistochemisty. Core B, serving as a GLP facility, will monitor immunologic responses to the administered vaccines by performing ELISPOT assays, tetramer analyses or cytokine flow cytometry (CFC). Cytokines in supernatants or body fluids will be monitored by the immunobead-based multiplex method established in the IMCPL. Core B will also be prepared to assist the SPORE investigators in implementing assays necessary for evaluation of immunologic responses to vaccines, including phenotypic and functional assays for regulatory T cells (Treg). The Core will ensure that all cellular products it generates and samples it collects are accompanied by appropriate documentation that will permit linking laboratory analyses with clinical results. Core B will also provide assistance in preparation of IND submissions. The Core laboratory has a long history of collaboration with all of the SPORE investigators, and in the context of the proposed clinical and pre-clinical studies will be entirely dedicated to the support of therapies being developed for patients with melanoma or cutaneous T-cell lymphoma (CTCL).
CORE C: Biostatistics Core CORE C (Biostatistics) will reside within the University of Pittsburgh Cancer Institute’s (UPCI) Biostatistics Facility, which provides clinical and basic-science investigators in UPCI with statistical expertise in design, analysis, and reporting of cancer-related research studies. These cover basic-science studies; phase I and phase II oncology clinical trials; epidemiologic studies, including those related to cancer prevention and awareness; and investigations of behavioral and health sequelae of cancer treatment. In its role as Core C for this Program Project, the Biostatistics Core will support all five research projects. We will collaborate with investigators on statistical aspects of the design of in vitro and in vivo laboratory-based studies as new data come to light, and perform both exploratory and confirmatory statistical analyses of the resulting data from key experiments in Projects 1 - 5. For the clinical trial that is planned (Project 4), we will collaborate with basic scientists and clinicians on further developing and finalizing a protocol that is methodologically sound, and that will pass scientific, ethical, and regulatory review. We will perform interim analyses of safety for the SPORE’s clinical trials (Projects 2, 3 & 4), and final analyses of their data on safety, immune response, and treatment efficacy. We will contribute to the review of the SPORE’s developmental research-program proposals, and provide statistical support to those that are funded. We will provide statistical support to the career-development awardees. We will work with the project investigators, with Core D (Informatics), and with UPCI Clinical Research Services, a component of Core A, to ensure that the requisite laboratory and clinical-trial data are available for statistical analyses. We will collaborate with the project investigators in writing and preparing progress reports, abstracts, manuscripts, and presentations.
CORE D: Informatics To optimally and efficiently support clinical and translational research, one must go beyond gathering data from researchers, and utilize the tools and expertise of modern informatics. The spectrum of needs, from those of a basic scientist to those of an outcomes-based clinical researcher requires a robust architecture of software, hardware, and information technology professionals to develop and maintain these tools. The cost of equipment, the rapid pace of technology development and the specialized expertise required to properly manage the informatics needs of the MSCP are not possible without high quality and efficient informatics services. It is therefore critical that this resource be well supported and integrated into the research mission of UPCI and the particular needs of the MSCP. The Informatics Core of the MSCP is composed of the following integrated services:
MSCP will be increasingly reliant on the provision of robust research information services, to more effectively support the translation of innovation from the laboratory to the bedside and to utilize critical clinical and outcomes data in the clinic to guide discovery at the bench. Dr. Becich, leader of this core, is also the Biomedical Informatics Core leader for the newly funded Clinical and Translational Science Institute at the University of Pittsburgh as well as the Cancer Informatics Core of the UPCI’s Cancer Center Support Grant (CCSG). This additional level of integration with NIH Roadmap Initiatives and the UPCI is a key strength of the proposed Informatics Core D of the Skin Cancer SPORE. |
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