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Vanderbilt University
To view the Vanderbilt Lung SPORE activities, please visit http://www.vicc.org/lungspore.
OVERALL ABSTRACT
Principal Investigator(s): David P. Carbone, M.D., Ph.D.
This Lung Cancer SPORE application is a new application submitted by the Vanderbilt-Ingram Cancer Center and its affiliated institutions. In this proposal, we apply the translational research strengths of the Vanderbilt-Ingram Cancer Center toward reducing the incidence, morbidity, and mortality of lung cancer, by focusing on the discovery and validation of molecular targets for prevention and therapy. In project 1 we are studying the role of specific matrix metalloproteinases and targeted inhibitors in the development and behavior of lung cancer. In project 2 we are applying sophisticated cDNA microarray and protein mass spectrometry techniques to the identification of molecular fingerprints of lung cancer. These fingerprints could ultimately be used to guide patient care or discover novel molecular targets for therapy. Project 3 studies a new potential molecular target, Notch3, that we identified by mapping a balanced chromosome translocation. This also represents a completely new mechanism for gene activation in lung cancer. Receptor tyrosine kinase inhibitors are an exciting new class of molecularly targeted reagents, and in Project 4 we study their effects on downstream signaling pathways and their use in combination with radiation therapy in anti-angiogenic tumor therapy. In projects 5 and 6 we investigate the role cyclooxygenase 2 (COX2) in the therapy (Project 5) and prevention (Project 6) of lung cancer. We have unique facilities for the analysis of COX2 metabolites and intend to study eicosanoid production by human lung cancer tumors in situ, its association with tumor angiogenesis, and its response to treatment with specific inhibitors in vivo. For Project 6 we propose to use two cohorts with previously collected pharmacy data to study the impact of long term COX2 inhibitors on the incidence of lung cancer. The first is a retrospective cohort study over 10,000 enrollees of the Tennessee Medicaid Program who were diagnosed with chronic obstructive pulmonary diseases (COPD) during the period of 1980 to 2002. The second is a population-based cohort and nested case-control studies of over 150,000 users of NSAIDs in North Jutland County, Denmark during the period of 1991 to 2002. In order to accomplish these research goals, we propose 4 cores: administrative, tissue, clinical, and biostatistical. The proposed career development and developmental research programs are tightly integrated with established institutional initiatives with documented track records of identifying and funding promising projects and individuals. We will use these established mechanisms to fund lung cancer-targeted career development and research projects. We believe that these projects, cores, and pilot and career development awards could lead to major improvements in the prevention, diagnosis, and treatment of lung cancer.
Project 1
Matrix Metalloproteinases in Lung Cancer
Principal Investigator(s): Lynn Matrisian, Ph.D.
Co-Investigator(s): Kenneth Hande, M.D.
Matrix metalloproteinases (MMPs) have been implicated in tumor growth, angiogenesis, invasion, and metastasis. There is extensive preclinical data that inhibition of MMP activity results in a reduction in tumor burden and prolongs the survival of treated animals. Synthetic MMP inhibitors (MMPIs) are currently in Phase III clinical trials in a variety of tumor types, and non-small cell lung cancer (NSCLC) trials in particular have been initiated with several different compounds. The results of the initial studies with the broad spectrum inhibitor marimastat (British Biotech) showed little benefit in advanced pancreatic and gastric cancer, but encouraging results in patients without overt metastases. Studies with the MMPI tanomastat (Bayer Corp.), however, were stopped when preliminary results from an adjuvant small cell lung cancer (SCLC) trial demonstrated that the MMPI was performing worse than placebo. These results raised the realization that very little is known about the role of MMPs in lung cancer development and progression. This proposal is designed to test the hypothesis that MMPs are valid therapeutic targets for lung cancer, and to determine the MMP family members that represent the most appropriate targets for MMPIs in NSC and SC lung cancer. The following specific aims are proposed: 1) Determine the expression profile of MMP transcripts and protein in SCLC and NSCLC samples and associate the expression profile with response to MMPI treatment. 2) Determine if MMPIs inhibit MMP activity in lung cancer patients using an ex vivo MMP activity assay, and 3) Determine the efficacy of MMPIs in chemically-induced and orthotopic preclinical models of lung cancer. The availability of mice that are null for several MMP family members provides the opportunity to examine the role of specific MMP family members in these model systems. These studies represent translational research that will guide the selection and application of selective MMPIs to the treatment of lung cancer.
Project 2
Molecular Fingerprinting of Lung Cancer
Principal Investigator(s): David P. Carbone, M.D., Ph.D.
Co-Investigator(s): Richard Caprioli, Ph.D.; Jason Moore, Ph.D.;
Mary E. Edgerton, M.D., Ph.D.
Lung cancer causes the deaths of more men and women in the United States than the next four most common types of cancer combined. Only a small fraction of cases are cured, and median survival is short. Some patients will die of metastatic disease, and some with only local disease. Some will respond to chemotherapy and some will not. While the molecular biology of lung cancer has been extensively studied, these variable outcomes cannot be predicted (or understood at a molecular level) by histopathology or currently available molecular markers. In this project, we intend to collect lung cancer tumor specimens from a national cooperative group clinical trial and utilize the unique combination of both protein and RNA-based technologies available at Vanderbilt to develop comprehensive molecular fingerprints of lung cancer. The goal is to determine molecular patterns that will be more predictive of tumor behavior, and may ultimately lead us to a better understanding of this behavior, improved interventions, and improved outcomes. The protein technology to be applied to this question utilizes novel microscopic laser-directed protein mass spectrometric analysis of tumor samples after laser capture microdissection. The same tumor samples will be used to probe large cDNA expression arrays, and the data from each analyzed for statistically significant signatures in defined sets of tumors with complete clinical follow-up. Analytical and statistical techniques will be developed as needed to analyze the data derived from these two technologies. Specifically, we will address Stage I node negative resectable non-small cell lung cancer (NSCLC) patients. In these samples we will identify molecular signatures of the following biologic behaviors: 1) occult lymph nodal involvement at the time of thoracotomy, 2) recurrence with metastatic disease vs. local recurrence, and 3) disease free and overall survival. This unique combination of clinical, technical, and analytical resources will be combined in this proposal to discern and evaluate molecular fingerprints of biology in lung cancer and these data may then lead to increased understanding of the basis of this biology and improved outcomes for patients with lung cancer.
Project 3
Chromosome Translocations and the NOTCH3 Signaling Pathway in Lung Cancer Biology
Principal Investigator(s): David P. Carbone, M.D., Ph.D.
Co-Investigator(s): Thao P. Dang, M.D.
Specific somatically acquired chromosomal translocations as a mechanism for oncogene activation are common in hematopoietic tumors and sarcomas, but have not been demonstrated in epithelial tumors. We have established a cell line, HCC2429, from an aggressive, metastatic lung cancer arising in a young woman with a minimal smoking history that had a normal karyotype except for a single translocation between chromosomes 15q and 19p. The cell line was established and grows exceptionally well and stably maintains this translocation as its only karyotypic abnormality, suggesting a potent transforming activity. We have mapped the breakpoint of this translocation on chromosome 19 to a site approximately 40 kb upstream from the start site of Notch3, a member of Notch proto-oncogene family not previously associated with cancer. This translocation is associated with massive overexpression of Notch3, supporting the hypothesis that the t(15;19) translocation results in the deregulation of this putative cellular proto-oncogene. We have preliminary data that about 25% of non-small cell lung adenocarcinomas express Notch3 and no squamous or small cell tumors. FISH has identified rearrangements in the 5Õ region of Notch 3 in at least some of these tumors as well. We have thus identified recurring specific translocation as a novel mechanism for oncogene activation in human lung cancer as well as a novel putative oncogene not previously known to be involved in human cancer. In this proposal, we will complete the molecular characterization of the identified translocations, determine the frequency and clinical significance of Notch3 receptor and ligand over-expression in lung cancer and normal tissues, and perform studies to characterize the transforming nature of Notch3 and its effects on downstream signaling pathways in lung cancer.
Project 4
FLK-1 Signal Transduction in Lung Angiogenesis and Radiation Biology of Lung Cancer
Principal Investigator(s): Dennis Hallahan, M.D., Ph.D.
Co- Investigator(s): Hak Choy, M.D.; Richard Caprioli, Ph.D.; Charles Lin, Ph.D.
We have identified a novel survival pathway within tumor vascular endothelium that enhances the therapeutic effects of ionizing radiation. Considering that radiation therapy is the primary modality of treatment for unresectable lung carcinoma, improving the efficacy of this treatment is important to improve the cure rate of lung cancer patients. In this regard, we have collaborated with several pharmaceutical companies that are developing small compounds that inhibit specific enzymes within the tumor endothelium. We will only investigate compounds that have entered or will enter clinical studies within the next 3 years. These compounds include SU5416 and SU6668 (Sugen) and PTK87/2K222584 (Novartis). These compounds inhibit receptor tyrosine kinases and downregulate the survival pathway within the tumor endothelium. These compounds will also enter clinical studies through the clinical core in this lung cancer SPORE application. An example of the success in this endeavor is the clinical protocol investigating SU5416 with radiation in a neoadjuvant lung cancer study. We anticipate additional studies with other compounds and future phase III trials. In addition to this translational approach, we will also investigate the mechanisms by which Flk-1 antagonists enhance the therapeutic effects of radiation. We will study the PI3 kinase and Akt/PKB signal transduction pathways. Our preliminary findings indicate that inhibition of this pathway enhances radiation-induced apoptosis in the vascular endothelium. We have entered into a material transfer agreement with ICOS, Inc. ICOS has identified several small compounds that inhibit specific isoforms of PI3 kinase. We will determine which of these compounds is most effective in enhancing the therapeutic effects of radiation in lung cancer models in mice. These compounds will then be developed at ICOS to enhance the pharmacokinetics and minimize toxicity. The same approach will be taken with Akt inhibitors as they are developed. In summary, findings from these preclinical studies will assist us in the design of phase I and feasibility studies in the clinic through the clinical core in this SPORE application. In addition, our close collaboration with pharmaceutical companies will ensure that these compounds move rapidly into clinical trials.
Project 5
Role of COX-2 in Clinical Lung Cancer Biology and Angiogenesis
Principal Investigator(s): David H. Johnson, M.D.
Co-Investigator(s): Jason Morrow, M.D.; Raymond DuBois, M.D., Ph.D.;
John R. Roberts, M.D.
Upregulation of cyclooxygenase-2 (COX-2) has been shown to be an early event in colon carcinogenesis. Multiple lines of evidence suggest that COX-2 upregulation is also an early event in the development of non-small cell lung cancer (NSCLC). In humans, COX-2 expression is upregulated in about one-third of atypical adenomatous hyperplasias and carcinoma in situ specimens obtained from lung, and in 70%-90% of invasive adenocarcinomas of the lung. The proportion of adenocarcinoma cells with increased COX-2 expression is much greater in lymph node metastases than in the corresponding primary tumors. Preclinical data indicate tumors with upregulation of COX-2 synthesize high levels of prostaglandin E2 (PGE2). High PGE2 levels are associated with increased production of proangiogenic factors and enhanced metastatic potential. These findings suggest that an increase in COX-2 expression may play a significant role in the development and growth of NSCLC and possibly with the acquisition of an invasive and metastatic phenotype. Specific inhibitors of COX-2 are now available and may prove useful in understanding the role of eicosanoids in lung cancer pathogenesis as well as in the management of established malignancies and possibly as chemopreventive agents. However, there are limited data on the function of tumor overexpression of COX-2 in lung cancer patients, and no data on whether selective inhibitors actually affect COX-2 activity within the targeted tumor in vivo. We propose to study the effects of specific inhibitors of COX-2 on COX-2 expression, serum VEGF levels and urinary metabolites of PGE2 in patients with lung cancer. Our results will serve as a prelude to clinical trials in which these agents are employed therapeutically. Our preliminary data suggest inhibitors of COX-2 rapidly reduce enzyme activity as determined by measurements of urinary metabolites of prostaglandins and assessment of enzyme activity within the tumor itself. These experiments will expand upon these preliminary results.
Project 6
Epidemiologic Studies of Lung Cancer Risk in NSAID Users
Principal Investigator(s): Wei Zheng, M.D., Ph.D., M.P.H.
Co-Investigator(s): Edward Arrowsmith, M.D., M.P.H.; William Blot, Ph.D.
Cumulative evidence fromin vitro and animal studies suggests that the enzyme cyclooxygenase-2 (COX-2) is important in the development and progression of lung cancer. Epidemiologic studies evaluating the association between the use of aspirin (an inhibitor of COX-2) and the risk of lung cancer have been conflicting, and no study has been conducted to evaluate non-aspirin non-steroidal anti-inflammatory drugs (NSAIDs). Using pre-recorded drug prescription databases of the Tennessee Medicaid program and North Jutland County of Denmark, we propose to conduct two studies in these populations to examine the effect of NSAID use on the risk of lung cancer. The first is a retrospective cohort study of over 10,000 enrollees of the Tennessee Medicaid Program who were diagnosed with chronic obstructive pulmonary diseases (COPD) during the period of 1980 to 2002. The second is a population-based, retrospective cohort study of over 150,000 users of NSAIDs in the general population of North Jutland County during the period of 1991 to 2002. Within the Danish cohort will be a nested case-control study of 350 cases and 700 controls, in which relevant information will be obtained on over the counter (OTC) analgesic use, as well as cigarette smoking and other potential confounding factors. The two studies proposed here complement each other and provide for an international comparison of NSAIDs as possible lung cancer chemoprevention agents. Because the data on NSAID use have already been collected, the studies will be very cost-efficient. More importantly, the use of pre-recorded pharmacy records minimizes potential errors in exposure assessment and provides a major advantage over existing cohort studies in evaluating the potential chemopreventive effect of NSAIDs. Given the high incidence and mortality of lung cancer and high prevalence of NSAID use, the results from our studies may have important public health implications in lung cancer prevention, and could set the stage for future randomized trials of COX-2 inhibitors in cancer prevention.
Administrative Core
Principal Investigator(s:) David P. Carbone, M.D., Ph.D.
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 reviewers.
Tissue Core
Principal Investigator(s): Joyce Johnson, M.D.
Co-Investigator(s): Roy Jensen, M.D., Ph.D.; John R. Roberts, M.D.
The tissue resource and pathology core will provide high-quality tissue specimens and basic histology and pathology services in support of lung cancer research performed within the SPORE. The specimens, information, and services provided by the core will be carefully quality-controlled, and informed consent and patient confidentiality will be consistently maintained. The primary function of the core will be to procure, archive, and distribute to SPORE investigators lung cancer tissue specimens and paired normal tissue which has been characterized histopathologically, along with relevant clinical data, if allowed by the specific protocol involved. Such tissue will be maintained in a variety of conditions to allow DNA, RNA, or protein studies, depending on the research requirements of individual investigators. The core will also provide laser capture micro-dissection technology. Data obtained from individual specimens will be maintained in the core's database. Establishment of the core functions will be facilitated by the prior existence of the Vanderbilt-Ingram Cancer Center's Human Tissue Acquisition and Pathology Shared Resource, established in 1993 and managed since its inception by Roy Jensen, MD, who is a co-PI on this core. The specific aims of this core are:
To collect, accession, and store tissues removed from lung cancer patients and normal ÒcontrolÓ lung as outlined in the appropriate research informed consent, and to preserve these tissues frozen, formalin-fixed and embedded, and on touch imprints, in order to support lung cancer SPORE projects.
To maintain a centralized, computerized database of all specimens with basic demographic and pathologic information to permit the integration of findings by SPORE investigator through molecular assays and other laboratory studies with risk factor data and follow-up.
To maintain the confidentiality and integrity of the database, through keyed numeric identifier assigned by the tissue core for each specimen.
To provide high quality control of all well-characterized tissues in both human and murine with respect to preservation and histopathologic characterization.
To provide micro-dissected lung tumor and matched control cell populations from paraffin and frozen section for appropriate projects (e.g. project # 2).
To provide immunohistochemical evaluation and quantitation. in both human lung and murine tissues.
Clinical Core
Principal Investigator(s): Alan Sandler, M.D.
The role of the Clinical Trials Core is to provide expertise in the development, implementation and coordination of all translational clinical trials resultant from the other SPORE projects that will ultimately lead to a better understanding of the biology of lung cancer. This improved understanding will ultimately lead to improvements in the treatment of lung cancer. To achieve this goal, the major responsibilities of the Clinical Trials Core will be 1) Provide expertise in the development and implementation of translational clinical trials related to the other SPORE projects 2)Accrual of patients to participate in SPORE initiated trials, 3) Timely and accurate collection of data, and 4) Accessibility of data for analysis by the various SPORE researchers at Vanderbilt university as well as researchers at other Lung SPORE sites. During the first year there will be at least seven translational trials open for accrual. These trials will serve to further the scientific knowledge regarding the role of 1) molecular fingerprinting and outcome analysis in resectable patients with lung cancer and 2) angiogenesis in lung cancer. More specifically, the impact of matrix metalloproteinases and their inhibition; the role of VEGF and its inhibition; the role of COX-2 and its inhibition on the outcomes of patients with lung cancer treated with either surgery, radiation, chemotherapy and/or their combination in patients with lung cancer. Over the five year course of the SPORE grant, trials will open and close. One of the goals of this SPORE is that the successful completion of these early pilot trials will ultimately lead on to larger scale, multi-institution trials for confirmation of our results. A second goal is that additional translational clinical trials will be designed based upon the results of the early pilot trials as well as the ongoing research of the participating SPORE investigators. The current pilot trials will thus serve as the template for further translational research in lung cancer.
Biostatistics Core
Principal Investigator(s): Y
Core IV Abstract - Biostatistics
The purpose of the Biostatistics Core is to provide professional expertise in statistics and database development for all Vanderbilt University Lung 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:
To provide study design and review all laboratory, animal and clinical studies
including feasibility assessment, power analysis and sample size estimation.
To collaborate in projects data analysis, interpretation of results, and the writing of final study reports and manuscripts.
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.
To work with Clinical Core in the development of research project database, to maintain data quality control and to ensure timely data capture.
To develop and evaluate statistical methods for experimental design and data analysis.
The Biostatistics Core support is required in all Lung Cancer SPORE studies. Core personnel has worked and will continue to work closely with project leaders for assuring that Core provides state-of-the-art statistical support.
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