|
|
||||
|
|
|
|
|
 |
 |
 |
 |
 |
 |
 |
 |
|
|
University of Texas M.D. Anderson Cancer Center
Overall Abstract Principal Investigator(s): Elizabeth A. Grimm, Ph.D. The overall goal of this University of Texas M.D. Anderson Cancer Center SPORE in Skin Cancer is to facilitate innovative research in the prevention, detection and treatment of melanoma leading to the elimination of this disease. This SPORE will achieve this goal by assembling a talented group of clinical and laboratory scientists who are committed to the translation of findings specific for melanoma from the lab to the clinic as well as from the clinic to the laboratory. The University of Texas M.D. Anderson Cancer Center has made as one of its priorities the elimination of melanomas as a health risk, and to this end, established in 1998 a Multidisciplinary Research Program in Melanoma to support training and research across traditional administrative boundaries in order to advance treatment of patients with melanoma. With this support, we have successfully organized an efficient and productive infrastructure that is the framework for this SPORE application. We are now poised to take advantage of the rapid increases in our understanding of melanoma at the molecular and cellular levels. Through this SPORE in Skin Cancer, we will enhance the translation of insights gleaned from research in melanoma biology and epidemiology to more effective prevention, detection and treatment approaches. This SPORE in Skin Cancer proposes five translational research projects, three career development positions and at least four developmental research awards all being served by three cores (Administrative; Informatics, Tissue Resource, and Pathology; and Biostatistics). The five projects address MHC-II-presented melanoma peptides engineered for optimal immunogenicity for CD4 cell activation (Project 1); molecular epidemiology of DNA repair enzyme function, genetics, and risk (Project 2); evaluation of inducible nitric oxide synthase as a prognostic factor for survival and exploration of signaling cascades for therapy (Project 3); regulation of IL-8 and MUC18/MCAM in combination with conventional chemotherapy as therapeutic approaches (Project 4); and evaluation of specific class II HLA alleles as prognostic factors for survival and evaluation of the role of associated cytokines in disease progression (Project 5). Through this research program and with the full support of the University of Texas, M.D. Anderson Cancer Center, we will make significant impact toward the prevention and detection of malignant melanoma processes, and treatment of patients with melanoma. Project 1 Studies in both humans and experimental models indicate that T cells play an important role in antitumor responses. Identification of MHC class I-restricted melanoma antigens has provided new opportunities to develop antigen-specific antitumor immunity. However, the clinical response elicited by such vaccines have been weak and transient, suggesting the need for CD4+ T cells to generate optimal antitumor immunity. Despite the recognized role of CD4+ T (helper) cells in antitumor immunity, relatively little is known about the MHC class II-restricted antigens recognized by tumor-specific CD4+ T cells, posing a major obstacle to the development of more effective cancer vaccines. The goal of this proposal is to identify and test MHC class II-restricted melanoma peptides that are presented on the cell surface for CD4+ T cell recognition in the context of HLA-DR3, and –DR13 molecules, which are expressed in more than 45% of the general population. The underlying hypothesis is that vaccination with MHC class II antigens, in combination with an effective MHC class I peptide, will stimulate tumor-specific CD4+ T cells, leading to more potent antitumor immunity than could be obtained using either peptide alone. Four specific research aims are proposed to test this prediction. Aim 1 will seek to identify new class II-restricted peptides through the use of recently established tumor reactive CD4+ T cell lines/clones and a novel cloning technology. The ability of these and previously identified MHC class II peptides to induce CD4+ T cells will then be enhanced by modifying key residues in T-helper peptides and delivering them into dendritic cells (DCs) (Aim 2). The immunogenicity of the most promising peptide candidates emerging from Aim 2 will be further evaluated by comparing their ability to elicit CD4+ T cell response from patient-derived peripheral blood mononuclear cells (PBMCs) to responses in an HLA-DR transgenic mouse model, using peptide-loaded DCs (Aim 3). Finally, in Aim 4 the immunogenicity, as well as safety and efficacy, of the candidate peptides will be subjected to clinical testing in melanoma patients, and to evaluation of the immunotherapeutic potential of a combined MHC class I and II peptide vaccine. The applicants’ record of success in the generation and testing of immunogenic tumor antigens, together with experience in clinical trials, bode well for further advances in this important area of cancer immunotherapy. Project 2 Using a case series of 600 patients with melanoma, we will perform an integrated molecular epidemiologic study to correlate genetic susceptibility phenotype and genotype data from lymphocytes and to extend these findings in surrogate tissue to the tumor tissue. We will measure functional nucleotide excision repair (NER) capacity by using the host cell reactivation assay with plasmids damaged by ultraviolet light and determine the frequencies of polymorphisms in the DNA repair genes (ERCC1, XPA, XPB, XPC, XPD, XPF, and XPG) implicated in the NER pathway. We will correlate the genotypes with functional NER activity. We hypothesize that individuals with “adverse” genotypes of the NER pathway will have poorer DNA repair capacity (DRC) than individuals with wild-type genotypes. In a subset of 400 patients, we will also determine the frequencies of mutations in the newly described BRAF gene and loss of heterozygosity (LOH) at 1p36, 6q22-23, 8p22-24, 9p21-22, 10q23, and 11q23 in tumor tissues and compare these tumor markers with sunlight exposure levels and constitutional markers of genomic instability (DRC and genotypes measured in lymphocytes). We hypothesize that the severity of mutations and loci-specific alterations in tumor tissues will be correlated with adverse susceptibility markers in surrogate tissue and with degree of exposure. The overarching goal of these studies is to determine whether the lymphocyte markers that we have already shown to be significant predictors of cancer risk (DRC and DNA repair gene polymorphisms) are an adequate reflection of genetic events in the target organ tissue. This finding will have substantial implications for future large-scale population-based molecular epidemiology studies and risk assessment for prevention and early detection of melanoma. Surrogate markers that best predict carcinogenic events in the target tissue will be useful for identifying high-risk subgroups for intensive screening and chemopreventive interventions. The ability to rapidly screen individuals for risk using minimally invasive procedures (blood analysis) has immense clinical implication. Project 3 Current data suggest that the process of melanoma tumorigenesis is enhanced by aberrant constitutive expression of the inducible form of nitric oxide synthase (iNOS), an enzyme catalyzing the generation of nitric oxide (NO), normally in response to cytokines. The presence of reactive NO in melanoma is indicated by detection of nitrotyrosine (NT), which is an end-product of the chemical reaction of NO with proteins; NT formation is known to alter intracellular signaling, especially when the target is a tyrosine kinase substrate. Production of low levels (pM) of NO by human tumors is proposed to be responsible for increased angiogenesis, growth, invasion, genomic instability and resistance to apoptosis. Our analysis of human melanoma specimens from patients with advanced (Stage III) disease indicates that patients whose tumors express iNOS are more likely to die within 2 years of neoadjuvant biochemotherapy than patients without iNOS (p<0.001). Specific Aim 1 comes from the clinic to the laboratory to further test the significance of iNOS and NT in tissue specimens as prognostic markers for primary and metastatic melanoma patients. The translational goals of Specific Aim 1 are validation of iNOS and NT as new prognostic markers for survival in melanoma patients and to determine whether either marker provides independent prognostic value. Significant results will then be submitted to American Joint Commission on Cancer, Committee on Melanoma for inclusion in future staging revisions, as the first molecular marker for melanoma to be validated. Specific Aim 2 tests the hypothesis that iNOS is controlled by specific, identifiable gene transcriptional regulators including constitutively active NFκB. NFκB activation is known to regulate basal as well as cytokine-induced iNOS in normal human cells, however its role in regulation of melanoma iNOS is unknown. Specific Aim 3 builds on further upstream mechanisms of iNOS activation and tests the association and functional role of mutated B-raf, which is reported to drive MAPK independently of Ras in melanoma cells. A novel molecular heteroduplex analysis will be employed to detect mutated B-raf in paraffin-embedded tumor specimens, and study of its association with iNOS and constitutive NFκB. The regulation of mutated B-raf activity is also proposed in Specific Aim 4 as a means to regulate iNOS and, by extension, melanoma growth. Project 4 Results from our laboratory have demonstrated that expression of Interleukin-8 (IL-8) by human melanoma cells correlates with their metastatic potential in vivo. Moreover, UV-B irradiation of primary cutaneous melanoma induces IL-8 mRNA and protein production and increases both tumor growth and metastasis in nude mice. In addition, ectopic expression of the IL-8 gene into primary cutaneous melanoma resulted in an increase in tumor growth and metastasis in vivo. We also demonstrated that IL-8 exerts its angiogenic effect by upregulating the expression and activity of MMP-2. These observations suggest that IL-8 could be a mediator of angiogenesis, tumor growth, and metastasis in melanoma and offered a potential target for immunotherapy against malignant melanoma. Indeed, using fully human neutralizing antibody to IL-8, (ABX-IL-8), as a single modality, we recently showed that it inhibited tumor growth and metastasis of human melanoma in nude mice. Since treatment of melanoma cells with the chemotherapeutic agent DTIC causes upregulation of IL-8, we reasoned that ABX-IL8 could potentiate the cytotoxic effect of DTIC. We now propose to evaluate the efficacy of combination treatment of ABX-IL8 plus DTIC in preclinical studies prior to the initiation of clinical trials in melanoma patients. Studies will also be performed to investigate the mechanisms by which IL-8 upregulates MMP-2 expression. MCAM/MUC18 is an adhesion molecule expressed on advanced primary and metastatic melanoma cells. MCAM/MUC18 contributes to the acquisition of the metastatic phenotype in melanoma by upregulating MMP-2 and increased attachment to vascular endothelial cells (extravasation). Therefore, blockade of MCAM/MUC18 might be a potential target to treat melanoma patients. We have recently developed a fully human anti-MUC18 antibody (ABX-MA1) and demonstrated that it can inhibit tumor growth, angiogenesis, and metastasis of human melanoma cells in vivo. Here we propose to examine the effect of cocktail treatment of ABX-IL8 + ABX–MA1 in preclinical studies prior to clinical trials in human. In addition, we will explore the possibilities of combining ABX-MA1 + DTIC and the RAF/MEK/ERK inhibitor BAY-439006 with DTIC. These studies may lead to the development of new modalities to treat patients with melanoma. Project 5 Polymorphic human leukocyte antigen (HLA) molecules (alleles) may influence melanoma progression through regulation of cytokine production and resulting T cell responses. Our previous work has shown that both specific HLA class II alleles and relatively high pre-operative plasma levels of IFN-γ are independent predictors of melanoma recurrence. In addition, one poor-prognosis HLA class II allele (HLA-DRB1*1101) is associated with both relatively high pre-operative IFN-γ levels and melanoma recurrence, suggesting that HLA class II alleles can regulate IFN-γ production in melanoma patients. Our preliminary in vitro data suggests that both peptide-specific as well as peptide-non-specific mechanisms can contribute to HLA class II allele-specific regulation of IFN-γ production. The goal of these studies is to determine how HLA class II alleles and altered cytokine production lead to melanoma progression.We hypothesize that specific HLA class II alleles influence melanoma progression by regulating cytokine production through both peptide-specific as well as peptide-non-specific mechanisms. In this project, we will determine if HLA class II alleles are prognostic markers in a large cohort of patients with early-stage melanoma (Specific Aim 1); we will evaluate a related set of potential prognostic markers mechanistically-linked to HLA class II and IFN-γ in a subset of this same patient population (Specific Aim 2); and we will evaluate potential allele-specific mechanisms regulating IFN- g production by measurement of in vitro IFN-γ production by HLA-typed melanoma patient lymphocytes (Specific Aim 3). HLA class II alleles, IFN-γ levels and the additional markers selected for investigation represent a group of mechanistically-linked, potentially important melanoma prognostic markers. Coordinated evaluation of these markers and their mechanisms will provide important information about their contributions to melanoma prognosis. These data will be incorporated into selection of patients for adjuvant therapies and used to prioritize markers selected for incorporation into upcoming clinical trials.
Core A The Administrative Core is critical to the success of the SPORE. The Core Leaders will facilitate functions of all Project and Core teams and stress the need for flexibility in scientific projects. Dr. Jeffrey Lee will chair the Executive Committee, which will be composed of one leader from each Project and Core. The Executive Committee will meet monthly to review scientific progress and fiscal status and will help surmount problems or barriers to assure that all goals are met within a realistic timeframe and within the budget constraints. The Administrative Core will schedule all meetings with investigators and assure optimum communication with investigators at M.D. Anderson Cancer Center and elsewhere. All SPORE investigators will participate in monthly scientific seminars to review research activities and will report bi-yearly to the Internal Advisory Committee. The External Advisory Committee will meet for one day annually to evaluate status and progress of all SPORE research programs. Specific responsibilities of the Administrative Core include
Coordination with other SPORE sites to promote and maintain communication and integration through sponsoring a yearly conference, and also through distribution of materials, electronic communications and evaluation of progress reports. Core B Effective procurement and utilization of well-characterized tissue is essential in any meaningful translational research. The Informatics, Tissue Resource, and Pathology Core (Core B) will work with each of the SPORE projects and with the Administrative and Biostatistics Cores to ensure maximum efficiency in the use of tissue for translational research directed at improving the prevention, detection, and therapy of melanoma. Core B will provide investigators at The University of Texas M. D. Anderson Cancer Center and other collaborating institutions with well-characterized, high-quality tissue, peripheral blood lymphocytes, plasma, and serum samples from patients with melanoma treated at M. D. Anderson Cancer Center whose clinical, pathologic, follow-up, and recurrence data are maintained in a comprehensive relational database. This SPORE facility will subsume the existing M. D. Anderson Cancer Center Melanoma Tissue Bank. Standardized procedures for procurement, processing, storage, quality control, histopathologic evaluation and distribution of samples will ensure optimal utilization and distribution of limited tissue samples according to the guidelines established by the Tissue Acquisition and Distribution Committee. A computerized Core database will track all samples from patient consent, to tissue acquisition and distribution of tissue and blood components to SPORE Projects. This system will contain comprehensive clinical information on all patients as well as relevant histopathologic characteristics for all samples and provide information on sample availability for future distribution through an NCI-sponsored tissue network. As over 900 new patients with melanoma are seen and treated at M. D. Anderson Cancer Center per year, this Core will be one of the largest available resources for translational research. The close relationship with the Biostatistics Core will allow an efficient analysis of the data produced by the different SPORE projects. Leadership for the Core is shared by a clinical investigator with expertise in melanoma patient care, database utilization and analysis of melanoma prognostic factors, and by a dermatopathologist with expertise in melanoma, including histopathologic evaluation, molecular analysis and quality-control procedures; this shared leadership will ensure maximal utilization of samples without compromising patient care. This centralized, comprehensive Core will contribute significantly to the success of the multidisciplinary and translational research projects outlined in this proposal. Core C The Biostatistics Core will provide support for the data management and analysis requirements of the projects and other cores of the SPORE. In addition to performing the planned analyses described in the descriptions of the individual projects, the Biostatistics Core will also assist in the design and analysis of future studies arising from the results of the proposed research and will facilitate the exchange of data and information between the various components of the SPORE. Because of the innovative nature of the research proposed and the high level of interaction between the various projects, the Biostatistics Core will play a vital role in the work of the SPORE by helping transmit results between Projects and rapidly developing new pre-clinical and clinical studies based on those results. UT M.D. ANDERSON CANCER CENTER SPORE IN MELANOMA Project 1 Rongfu Wang, Ph.D. Yukiko Kiniwa, Ph.D. Adrian Gee, Ph.D. Linh Xuan Doan, Ph.D. Project 2 Qingyi Wei, M.D., Ph.D. Li Mao, M.D. Sara Strom, Ph.D. Julie Ellerhorst, M.D., Ph.D. Madeleine Duvic, M.D. Project 3 Elizabeth A. Grimm, Ph.D. Merrick Ross, M.D. Suhendan Ekmekcioglu, Ph.D. Project 4 Menashe Bar-Eli, Ph.D. Luis Camacho, M.D., M.P.H. Project 5 Jeffrey E. Lee, M.D. John D. Reveille, M.D. Constantin Ioannides, Ph.D. Core A Elizabeth A. Grimm, Ph.D. Jeffrey E. Lee, M.D. Core B Jeffrey Gershenwald, M.D. Victor Prieto, M.D., Ph.D. Core C Lyle Broemeling, Ph.D. Marcy Johnson, M.S. |
 |
|||
|
|