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Department of Medicine at Vanderbilt University Medical Center, Nashville, TN
VANDERBILT SPORE IN GI CANCER This Gastrointestinal SPORE application was submitted by the Vanderbilt-Ingram Comprehensive Cancer Center 9VICCC) and its affiliated institutions. This proposal is from a group of basic science researchers and clinical investigators with long-standing collaborative interactions that have resulted in significant NCI extramural funding. In this proposal, we apply the translational research strengths of the VICCC and its affiliated institutions towards reducing the incidence, morbidity, and mortality of colorectal cancer by focusing on established (EGF receptor and cyclooxygenase-2), promising (p120) and to-be-identified molecular targets for prevention and therapy. We propose five projects. In project 1, we will examine molecular correlates of EGF receptor blockade in an ECOG-approved Phase II trial of ZD 1839. In parallel with this, we will use polarizing colon cancer cell in vitro to evaluate ways in which blockade of the EGF receptor axis can be optimized. In project 2, we will evaluate combined blockade of the EGF receptor signaling pathway and cyclooxygenase-2 in Phase I and, subsequently, Phase II trials in patients with colorectal cancer. These trials will be complemented by genetic studies to examine the importance and interaction of these two pathways in three mouse models of intestinal cancer. Project 3 is designed to utilize DNA microarray and imaging mass spectrometry to identify and, ultimately, predict rectal cancer patients most likely to respond to neoadjuvant chemoradiotherapy. Project 4 will examine the role of p120 - a protein first identified by Vanderbilt investigators - in colorectal metastasis. Project 5 is designed to test and identify markers for adenoma recurrence that could eventually serve in the early detection or prevention of colorectal cancer. To support these research projects, we propose six cores: administrative, tissue, clinical trials, emerging technologies, biostatistics and biomedical informatics. The proposed developmental (pilot) research and career development programs are tightly integrated with established institutional initiatives that have documented track records of identifying and funding promising projects and individuals. We will use these established mechanisms to fund gastrointestinal cancer-targeted pilot projects and to support career development. The strong institutional commitment from both VICCC and Vanderbilt University Medical Center to the success of this program is outlined in the application and supporting documents. We believe that the projects and career development awards outlined in this application will lead to major improvements in the prevention, diagnosis, and treatment of colorectal cancer. Project 1. EGFR Axis as a Therapeutic Target in Colorectal Cancer The epidermal growth factor receptor (EGFR) is overexpressed in more than 50% of colorectal adenocarcinomas and is associated with a more aggressive and invasive phenotype. We have received approval from the Cancer Therapy Evaluation Program (CTEP) of NCI to conduct a randomized Phase II trial of ZD1839 (Iressa), an oral EGFR receptor tyrosine kinase inhibitor (EKI), in patients with recurrent or refractory colorectal cancer (CRC) through the Eastern Cooperative Oncology Group (ECOG). We will obtain pre- and post-treatment tumor biopsies in a subset of patients to assess changes in downstream effectors of EGFR activation. We hypothesize that ZD1839 will reduce EGFR-mediated signaling events. Preclinical in vitro and in vivo studies will be performed in parallel with this clinical trial to elucidate the mechanisms by which ZD1839 and related compounds exert a growth inhibitory effect and to identify other factors that may influence blockade of the EGFR axis. It is likely that the site of blockade of the EGFR axis (e.g., cell surface release of ligand, ligand binding to EGFR, intrinsic tyrosine kinase activity of receptor, or even EGFR RNA production) will confer certain biological effects that are different than agents that block the EGFR axis or its signaling at another point in its cascade. The goal of this project is to characterize the molecular and clinical events associated with blockade of the EGFR axis using small molecules and monoclonal antibodies. The results from these preclinical studies will be used to design follow-up clinical trials and tissue correlative studies. The following specific aims are proposed: (1) To identify molecular markers of EGFR inhibition from tumor tissue obtained just prior to and 1 week after the initiation of ZD1839 in patients treated on an ECOG Phase II trial. (2) To elucidate mechanism(s) by which ZD1839 and other EGFR tyrosine kinase inhibitors influence the growth of two human CRC cell lines (HCA-7 and HCT-116) in nude mice. (3) To utilize polarizing CRC cells in vitro to identify other factors that interact with EGFR and its signal transduction pathway in order to optimize the pharmacological and biological blockade of the EGFR axis. Project 2: Combined Blockade of EGF Receptor and COX-2 in Intestinal Tumorigenesis A major hypothesis of this GI SPORE is that EGF receptor (EGFR) and cyclooxygenase (COX-2) are critical participants in the pathogenesis of colorectal cancer (CRC). We will employ mouse models to further explore the role of these two pathways in the pathogenesis of intestinal neoplasia, in addition to advancing the use of pharmacological blockade of EGFR and COX-2 in patients with advanced CRC. Abnormal EGFR signaling appears to be a necessary step in the early development of intestinal tumors in ApcMin mice, a mouse model for intestinal neoplasia caused by a null allele of the Apc gene. We have recently demonstrated that when ApcMin mice are crossed to Egfrwa2 mice (a hypomorphic mutant in the Egfr gene resulting in substantially reduced EGFR activity), a 90% reduction in tumor number, but not tumor size, occurs by the time the mice are 3 months old. Previously, it has been reported that when ApcD716 mice are crossed with Ptgs2tm1Jed (Cox-2 null mice), an 80% reduction in tumor number is observed at 3 months of age and the size of the remaining tumors is also reduced. We propose to study the interaction of these two pathways during intestinal tumorigenesis. Our hypothesis is that the EGFR and COX-2 pathways act in a complementary manner. We predict that blockade of both pathways will result in a more complete reduction in tumor formation in murine models of intestinal neoplasia. We further predict that combined use of drugs that block EGFR tyrosine kinase and COX-2 will be an effective strategy for treatment of patients with recurrent CRC than either drug alone. In Specific Aim 1, we will examine tumor formation in three complementary mouse models - spontaneous small intestinal tumors (ApcMin mice), spontaneous colon tumors (Smad-3 null mice), and carcinogen-induced colon tumors (azoxymethane [AOM]-treated A/J mice) - in which the Egfr and Ptgs2 genes have been disrupted. In Specific Aim 2, we will refine specific assays to evaluate the effects of pharmacologic blockade of EGFR tyrosine kinase and COX-2. In Specific Aim 3, we will conduct a Phase I clinical trial that combines an EGFR tyrosine kinase inhibitor with a selective COX-2 inhibitor to determine the tolerability of this combination that will lead to a Phase II trial to evaluate the clinical activity and biochemical impact of combined EGFR/COX-2 blockade on patients with advanced CRC. Project 3. Molecular Profiling of Rectal Cancers to Evaluate the Role of COX-2 Adjuvant chemotherapy and radiation therapy have had a modest effect on recurrence rates and survival for patients with node positive or transmural (stage II/III) rectal cancer, but approximately half of these patients will suffer a recurrence and most of the patients who recur will die of their cancer. Following surgical resection, a small but significant minority of patients with early stage rectal cancer experience lethal recurrences of their tumors. For this subset of patients, adjuvant therapy has, to date, shown no significant benefit. Increased expression of cyclooxygenase-2 (COX-2) occurs in approximately 80% of colorectal cancers, and several lines of evidence suggest that this increased COX-2 expression may promote tumor progression through increased tumor cell survival, increased invasiveness and increased tumor-associated angiogenesis. COX-2 inhibitors have shown promise in the prevention of the progression of colorectal polyps. In addition to a chemopreventive role, results of preclinical studies with selective COX-2 inhibitors also suggest that these new and nontoxic pharmaceuticals may have a role in the treatment of colorectal cancer. Experimental studies have also suggested that COX-2 inhibitors appear to enhance the effect of radiation therapy selectively on those tissues that express COX-2. The effect of treatment with COX-2 inhibitors on human colorectal cancer cells in vivo is unknown, as are the effects of these agents in combination with standard chemotherapy and radiation therapy. This project aims to examine the biological responses to a COX-2 inhibitor and the combination of a COX-2 inhibitor with chemoradiation in the treatment of rectal cancer. Rectal cancer was selected as a target tissue in this study because of the accessibility of the tumor tissue for repeated biopsy during a simple proctoscopic procedure that does not require sedation or mechanical bowel preparation beyond a pre-procedure enema. Specific aim 1: To determine VEGF expression and eicosanoid levels in tumors and in normal rectal tissues and to assess the effects of COX-2 inhibition on (1) Eicosanoid metabolism in both normal and tumor tissue, (2) VEGF expression and (3) Microvascular density. Specific Aim 2: To assess the effects of COX-2 inhibition on surrogate markers of eicosanoid expression. Specific Aim 3: To evaluate protein and RNA expression profiles pre and post administration of Celecoxib. Specific Aim 4: To determine whether changes in COX-2 expression, eicosanoid production (tissue/urine), gene and protein expression following celecoxib and celecoxib/RT can be used to predict clinical outcome in Stage II/III rectal cancer. Project 4. p120 Dysfunction in CRC Tumor progression in CRC involves accumulation of genetic and epigenetic changes that ultimately lead to malignancy. E-cadherin downregulation occurs frequently and is widely believed to be a pivotal event in the transition to metastasis. Interestingly, studies of human GI tumors report p120 loss in some cases, and dramatically upregulated p120 in others. In cultured cells, p120 overexpression induces significant changes in morphology and increases in cell motility, apparently through modulation of rho GTPases. We believe that the same mechanism drives the metastatic phenotype in E-cadherin deficient carcinomas, where p120 is stranded at high levels in the cytoplasm. Conversely, loss of p120 function in E-cadherin-positive cells appears to disrupt E-cadherin function by a different mechanism. Our preliminary observations suggest that the colon carcinoma cell line SW48 contains mutations in both alleles of the p120 gene and restoring wild type p120 expression induces a striking correction of the epithelial phenotype. Thus, under normal circumstances, p120 function is probably necessary for E-cadherin's tumor suppressor role. Furthermore, on COLO205 cells, E-cadherin malfunction is induced by aberrant signaling through a p120-dependent pathway, suggesting that cadherin function can also be disrupted by post-translational regulation of p120. These observations suggest roles for p120 in the genesis and/or progression of CRC. Through the specific aims summarized below, this proposal seeks to understand the significance of defects in p120 expression and signaling in human colon oncogenesis and to test in a mouse model the efficacy of inhibiting metastasis by blocking p120 function in cells that have lost E-cadherin expression.
Project 5. Epidemiologic Study of Predictors for Adenoma Recurrence Most colorectal cancers arise from adenomatous polyps and a large proportion of adenoma patients will develop new adenomas after their initial polypectomy. There is considerable controversy regarding an appropriate surveillance strategy for adenoma patients following the removal of their initial adenomas. Therefore, studies assessing predictors for recurrent adenomas, particularly among patients with multiple or pathologically advanced adenoma(s), will provide valuable information for designing individualized, cost-effective surveillance and chemoprevention strategies for adenoma patients. Some tumor markers (genetic or epigenetic alternations) involved in the formation of colorectal neoplasms may be promising predictors for recurrent adenomas, as they are believed to reflect a field cancerization process or a genetic predisposition to colon adenomas. We hypothesize that patients whose initial adenomas have certain altered genetic or epigenetic profiles may have an elevated risk of adenoma recurrence, and these tumor markers, along with pathologic features of initial adenomas can be used to predict the risk of adenoma recurrence. To evaluate these hypotheses, we propose in this application a series of investigations, consisting of both hypothesis-testing and hypothesis-generating components as described below. For the reasons described in section B1, the major focus of this application will be on the study of predictors for recurrent adenomas among patients with multiple or pathologically advanced adenoma(s). CORE I: ADMINISTRATIVE CORE The Administrative Core is responsible for managing the SPORE resources and facilitating communications between the SPORE components, other collaborators, and with other SPORES and the NCI. This is accomplished through a series of oversight committees, organized administrative and scientific meetings of SPORE investigators, institutional representatives, and external advisors. CORE II: TISSUE CORE The specific aims of this proposal are (1) To collect, process, bank, and distribute human colorectal neoplasms and matched normal tissue samples to investigators in the VUMC GI SPORE and other GI SPORES; (2) To collect and bank biopsies of grossly normal mucosa and mucosa adjacent to adenomas to facilitate research in adenoma recurrence; (3) To perform quality control to ensure that the relevant tissue is supplied to the researcher, and that tissues are suitable for the planned research (not necrotic or involved by unsuspected disease processes); (4)To protect patient confidentiality through use of an explicit pre-surgical consent form that specifically addresses use of extraneous tissue for research purposes and through de-identification of specimens, or through anonymization of specimens if informed consent is waived; (5) To work with the Biomedical Informatics Core to establish an informatics strategy for networking of requests, specimen tracking, and for extraction of de-identified data relating to specimens of interest. The Biomedical Informatics Core will help identify suitable specimens from the pathology archives, either by diagnosis, histologic features, demographics, clinical features, or other outcomes data, and will develop and maintain a centralized database of prospectively collected and banked specimens; (6) To provide laser microdissection services to the GI SPORE investigators; (7) To provide tissue microarray services to the GI SPORE investigators; 8) To provide correlation of tumor morphology with image mass spectroscopy findings (9) To provide expertise in evaluation of histopathology of mouse models of colorectal neoplasia and correlation with human disease; (10) To provide expertise in developing, performing and evaluating immunohistochemical stains for GI SPORE investigators. Establishment of a GI SPORE Tissue Core at VUMC will build upon two already established mechanisms for tissue collection: the Tissue Acquisition Shared Resource of the Vanderbilt-Ingram Cancer Center and the Tissue Collection Core for the Program Project Grant Molecular Mechanisms of Chemoprevention of Colorectal Cancer by NSAIDS (hereafter referred to as the COX-2 PPG), and a third mechanism in development, the newly-awarded VUMC- led site for the Cooperative Human Tissue Network (CHTN). Mechanisms for collection of human GI tissue and quality assurance are already in place and are used on a daily basis. By building upon the infrastructure of the VUMC CHTN, which collects tissue at three additional hospitals, a large number of GI specimens will be captured that would otherwise be unavailable for the GI SPORE.
CORE III: CLINICAL TRIALS CORE The specific aims of the Clinical Trials Core are (1) to assist investigators in the development and implementation of clinical translational trials conducted by projects within the SPORE; (2) to assist clinical investigators in the conduct of those clinical trials; (3) to ensure timely, accurate, and thorough collection and entry of clinical data into database management systems created and maintained by the Biomedical Informatics Core; (4) to monitor and assure the safety of research subjects, adherence to institutional and federal regulatory requirements, and compliance with protocol-specified activities; (5) to provide information, both on request and on an outreach bases, to patients, families, and health care professionals regarding clinical trials being conducted under this and other GI SPOREs; and (6) to monitor inquiries and track accrual to SPORE clinical trials. Establishment of a Clinical Trials Core will build upon already established mechanisms for clinical trial support at Vanderbilt including the Clinical Trials Shared Resource and the Clinical Protocol Review and Monitoring Committee of the Vanderbilt-Ingram Comprehensive Cancer Center, the Vanderbilt-Ingram Comprehensive Cancer Center Affiliate Network, and the Institutional Review Board of Vanderbilt University. The creation of a Clinical Trials Core within this GI SPORE grant is made necessary by the intensive needs and specialized nature of the clinical translational research projects described in this application. CORE IV: EMERGING TECHNOLOGIES CORE The Emerging Technologies Care consists of two components - a Microarray Core and a Mass Spectrometry Core. Microarray technology, which facilitates measurement of relative levels of gene expression through a massively parallel approach, has begun to revolutionize biomedical research. While microarray analysis measures relative mRNA abundance, mass spectrometry allows high-resolution analysis of protein levels. Emphasis in the GI SPORE Emerging Technologies Core is on providing high-density gene expression microarray and mass spectrometry technologies and support for their use. Initially, Project 3 (utilization of both microarray and mass spectrometry) and Project 5 (utilization of only microarray) will be supported. It is anticipated that these technologies will be utilized by other GI SPORE investigators during the GI SPORE granting period. The Emerging Technologies Core, operating in conjunction with the Vanderbilt Microarray Shared Resource (VMSR) and the Mass Spectrometry Research Center, will provide GI SPORE members affordable access to these technologies. CORE V: BIOSTATISTICS CORE The purpose of the Biostatistics Core is to provide professional expertise in statistics for all Vanderbilt University GI Cancer SPORE projects, investigators and participants. Functions provided by this core include development of experimental designs, data acquisition and database development, statistical analysis and interpretation of findings, and collaboration on presentation of results. To achieve these functions, the core director and core biostatisticians are constantly available to investigators, and are in regular contact with the project and core leaders. The primary objectives of the Biostatistics Core are (1) To provide study design and review all laboratory, animal and clinical studies including feasibility assessment, power analysis and sample size estimation. (2) To collaborate in project data analysis, interpretation of results, and the writing of final study reports and manuscripts. (3) To provide relational database design, data entry, data tracking, forms, queries, and reports, and to maintain computer databases for information storage and retrieval for all projects. (4) To work with Clinical Core and Bioinformatics Core in the development of research project database, to maintain data quality control and to ensure timely data capture. (5) To develop and evaluate statistical methods for experimental design and data analysis. The Biostatistics Core support is required in all GI Cancer SPORE studies. Core personnel have worked and will continue to work closely with project leaders to assure that the Core provides state-of-the-art statistical support. CORE VI: INFORMATICS CORE The purpose of the Biomedical Informatics Core is to provide professional expertise in informatics to enable interpretation of results generated from high throughput technologies in the post-genomic processing. The concept of a Biomedical Informatics Core extends beyond the scope of a traditional service core and builds upon the model set by Biostatistics Cores. Collaborative efforts with experimental investigators are covered by salary and equipment for the Core. This model stems from the very nature of the work being performed. Tissue informatics at the level described here is based on translation from current methods in de-identification, ongoing development in common data elements and controlled vocabulary, and is coupled with ongoing development of ontologisms. Similarly, analysis of high throughput post-genomic experimental data is a relatively new field without a proven gold standard. Finally, there is no method in existence for directly comparing gene expression array data and tissue mass spectrometry results. The methods to address these problems are being developed as a part of the Core activities. This model reflects a new paradigm for interactions that bridge between a Core and a Program with the intent of translating technology for the exploration of high throughput post-genomic data generated by the Emerging Technology Cores. Developmental Research Programs In an effort to promote excellent translational gastrointestinal cancer research, the Developmental Research Program is a major focus of this GI cancer SPORE application. The support of pilot projects allows initiation of research to move interesting basic science initiatives toward clinical implementation and provocative clinical observations to the lab. Solicitation and review of applications for pilot projects will be carried out jointly with the Vanderbilt-Ingram Comprehensive Cancer Center (VICCC), which conducts a very successful pilot project program funded by the Cancer Center Support Grant (CCSG). In fact, this component of the VICC-CCSG renewal application was judged as outstanding on the last competitive renewal. Utilization of the extant CCSG solicitation and review mechanism allows the SPORE to save the fixed costs of administering the program. This SPORE requests $150,000 per year for the support of pilot projects. The projects will be funded at the level of $50,000 for one year, with a one-year extension allowed after re-application. Career Development Vanderbilt University Medical Center and the Vanderbilt-Ingram Comprehensive Cancer Center have become increasingly aware of the reduced quantity and quality of postdoctoral training in translational research available to physician scientists (even M.D./Ph.D.) in comparison to full-time Ph.D., basic scientists, who hold the same academic rank. Not only is there an alarming shortage of physician-scientists but also the number of NIH-funded research initiatives targeting GI cancer is relatively small compared to the magnitude of the clinical GI cancer problem. The goal of this Career Development Program is to encourage and to prepare investigators for independent careers in GI cancer research. In some situations, more established investigators who wish to change their research direction to GI cancer may be supported. This GI cancer SPORE will support GI cancer-specific career development through three established program at Vanderbilt: 1) the Vanderbilt Physician Scientist Development (VPSD) Awards Program; 2) the Masters of Public Health (MPH) program and 3) the Masters of Science Clinical Investigation (MSCI) Program. |
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