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Johns Hopkins University
The Johns Lung Cancer SPORE is in its tenth year and continues to have as its goals, the performance of highly translational research to move ideas from the bench to bedside, and vice versa, to provide new means for the prevention of, risk assessment for, early detection of, gauging prognosis of, and therapy for, lung cancers of all types. In these efforts, extensive formal collaboration with other Lung Cancer SPORES is often emphasized. The program uses the flexibility of the SPORE funding mechanism to extend projects that continually evolve higher and higher translational potential and curtail those that do not. There is an emphasis on constantly bringing in new concepts and directions in parallel with fully evolving those areas that are headed for ultimate translational verification and even reaching common clinical practice. In terms of research at the highest translational level, including work at the population level, the SPORE is presently emphasizing two projects, Projects 2 and 3 (a collaborative venture with the Colorado SPORE), which are testing epigenetic molecular markers which appear to have very high promise for the areas of risk assessment, early detection, and gauging of prognosis of lung cancer. A third project, Project 1, also aimed at predicting lung cancer risk and prognosis, is taking new approaches to develop genetic markers for these purposes. A fourth project, Project 4, is developing new concepts for lung cancer prevention by taking the concepts through pre-clinical models and initial proof of principle studies in high risk individuals. This work is very complementary with participation of the Hopkins SPORE in the consortium chemoprevention trials (Lung Cancer Biomarker Chemoprevention Consortium-LCBCC) with the other Lung Cancer SPORES and with a collaborative trial of Iloprost in collaboration with the Colorado SPORE. Projects 5 and 6 are both aimed at development of novel therapeutic strategies for lung cancer with one exploiting new insights into these diseases derived from study of molecular pathways guiding early lung development and the other exploring inhibition of fatty acid synthesis as a new approach.
Project 1 Lung cancer is the leading cause of cancer related deaths in males and females in the United States. Surgical resection remains the only curative therapy for patients with non-small cell lung cancer (NSCLC). Identifying high-risk patients likely to develop cancer within a defined period of time is a clinical and biologic challenge for improving survival in patients with NSCLC. This study aims to use the power of molecular biology to develop and test promising new molecular markers for the identification of patients at risk of developing non-small cell lung cancer. Molecular studies have shown that clonal genetic alterations, e.g. 9p21 deletions, are often present in the lung epithelia of smokers and patients with a previous lung malignancy. This study will examine lung epithelium in smokers with a previous history of lung cancer and controls for the presence of specific chromosomal losses using state of the art techniques. New molecular markers will be developed and confirmed by genome wide SNP microarray analysis in normal appearing, preneoplastic, and malignant lung epithelium. The identified genetic markers will be used to test mucosa at risk in a unique population and develop a profile of genetic changes that predict progression to invasive cancer over a defined period of time. Genetic alterations at critical chromosome loci have been shown to be able to predict the progression of oral precursor lesions to invasive cancer. Our studies will pave the way for the development of similar markers in lung cancer and rapid translation into the clinical setting. Project 2 Lung cancer is the most common cause of cancer death in the United States, accounting for more life lost than breast, prostate, colon and rectal cancer combined. Of the estimated 190,000 individuals who are diagnosed each year, over 180,000 succumb to the disease. Increasing insight into the molecular basis of lung cancer pathogenesis offers hope to combat this disease. Lung cancer development and progression involves the inactivation of tumor suppressor genes and activation of oncogenes. While the accumulation of genetic alterations has been shown to be involved in the progression of lung epithelial cells from hyperlasia, metaplasia, dysplasia, carcinoma in situ, invasive carcinoma, and finally metastatic carcinoma, recent work in the previous funding period of this SPORE project has demonstrated that epigenetic changes represent another important molecular change in lung cancer. With that background, the specific aims of the current proposal are: Specific aim 1. To utilize a newly derived microarray approach to identify novel hypermethylated genes which will help comprise methylation marker panels providing for full coverage of the non-small cell lung cance genome. Specific aim 2. To utilize the marker panels from specific aim 1 to develop an epigenetic progression model based upon studies of precursor lesions and early stage lung cancer. Specific aim 3. To test the epigenetic marker panels for their efficacy as prognostic markers to identify patients with Stage I non-small cell lung cancer at very high risk for rapid disease recurrence.
Project 3 Collaborations between Drs. Belinsky and Herman (SPORE Career Development Recipient) over the past 5 years have been successful in defining the role of genes inactivated through aberrant methylation in the development of lung cancer. By increasing the sensitivity of a PCR approach for detecting methylated DNA sequences, we have demonstrated that aberrant methylation of the p16 and/or MGMT promoters can be detected in DNA from sputum in patients with squamous cell lung carcinoma (SCC) up to 3 years prior to clinical diagnosis. These findings suggest that aberrant gene methylation could be highly sensitive as molecular markers in propulation-based screening for early detection of lung cancer and as potential monitors to assess chemoprevention interventions. These findings led to the establishment of a new project with our SPORE in January 2001. This project has established a strong collaboration with scientists within the Colorado SPORE. Subjects and samples for this project will be derived largely from the University of Colorado Cancer Center Sputum Cytology Screening Cohort Study. This is an ongoing prospective study initiated in 1993 to determine whether exfoliated sputum containing cytological atypia predicts future lung cancer development in subjects with airflow limitation (COPD) and a history of heavy smoking. To begin developing a panel of methylation markers for predicting lung cancer risk, a nested, case-control study comparing sputum samples from 33 incident cases and their matched controls was conducted. The presence of any of four methylation markers examined was associated with a 6.3-fold increase in the risk for lung cancer. Moderate atypia or worse in sputum was also associated with a 4.1-fold increase in the relative risk for lung cancer over this time period. Interestingly, the methylation markers and cytology were not highly correlated with each other, though each was predictive of lung cancer risk, hence the two biomarkers were synergistic in conveying a 13.8-fold increase in relative risk. Through this cohort and a Phase II chemoprevention trial, studies with appropriate power will be designed to test specific hypotheses related to prediction of cancer risk and monitoring of chemoprevention interventions. This project is an inter-SPORE collaboration with Colorado that links clinical and epidemiologic findings with the development of promoter hypermethylation as molecular markers through the following three specific aims. Specific aim 1 will conduct a nested, case-control study within the Colorado cohort to evaluate longitudinally the ability to detect in sputum genes inactivated by methylation as biomarkers for predicting lung cancer risk either alone or in combination. Specific aim 2 will examine the dynamics of the field cancerization process by determining the concordance between methylation changes detected in sputum and bronchial biopsies from the same subject. Specific aim 3 will determine whether a panel of methylation markers can be used to predict the efficacy of the chemopreventive agen Iloprost in a randomized Phase II study through evaluation of bronchial biopsies and sputum collected at study entry and following completion of the intervention. * This is a collaborative effort between The Johns Hopkins University, Lovelace Respiratory Research Institute and Colorado Pulmonary Associates, P.C.
Project 4 This new SPORE project seeks to take promising laboratory leads for lung cancer prevention, beginning with animal models, to proof of principle clinical studies. Our first efforts will explore electrophiles generated after metabolic activation of chemical carcinogens as well as reactive oxygen species. Both are major causes of malignancy. Cancer chemoprevention by induction of protective phase II proteins to counteract the effects of these carcinogens has gained considerable attention. Isothiocyanates have proved to be potent inducers of phase II proteins and compelling epidemiological evidence suggests that dietary isothiocyanates are linked with decreased incidence of lung cancer. Deciphering the downstream targets of isothiocyanates can help in developing these compounds for cancer chemoprevention. The genes for phase II proteins contain the antioxidant or electrophile response element (ARE), which regulate their basal and/or inducible expression. Nrf2, a member of the basic leucine zipper family plays a central role in activation of these genes by binding to ARE in response to its activation by chemopreventive agents. Our studies have shown that Sulforaphane, a naturally occurring isothiocyanate, acts as a potent activator of Nrf2. In this work, we used a microarray approach to identify Nrf2 targets in intestine which included enzymes that detoxify a wide spectrum of electrophiles and tobacco specific carcinogens. The strategy of activation of Nrf2 for induction of phase II proteins recently has been shown to be effective among former smokers in a phase II b trial using anethole dithiolethione in lowering progression of pre-existing dysplastic lesions and appearance of new lesions. This proposal will focus on the hypothesis that activation of Nrf2 in lungs by Sulforaphane can lead to protection against lung cancer with the ultimate goal of developing this agent for clinical trials. The downstream targets of Nrf2 activation in lungs, identified using a genomics approach, will serve as biomarkers to monitor the efficacy of Sulforaphane for lung cancer chemoprevention in the NNK inducible A/J mouse lung cancer model. A small clinical trial will evaluate the efficacy of broccoli sprout extract, optimized for high amount Sulforaphane, to activate the Nrf2 pathway in individuals at high risk for lung cancer. Success in these studies will justify larger controlled studies in current and former smokers.
Project 5 Lung cancer results in more deaths than colon, breast and prostate cancer combined. Conventional cytotoxic therapy of lung cancer is limited by side effects, and is rarely curative. Mechanism based therapies directed at tumor specific pathways offers hope for the development of novel treatments. With this in mind, we have studied activation of mammalian development pathways in human lung cancer to provide insights into how such interventions can be achieved. The morphogen sonic hedgehog (Shh), which signals to adjacent embryonic cells to specify morphogenic patterns and progenitor cell fates, is essential for lung development. In extensive preliminary studies, we provide compelling evidence that many human lung cancers activate Hedgehog (Hh) signaling. We demonstrate cell autonomous Hh signaling in small cell lung cancer (SCLC), whereas non-SCLC (NSCLC) sends a Shh signal to adjacent stromal cells. Moreover, we find that specific inhibition of Hh signaling by the Veratrum alkaloid cyclopamine inhibits the growth of SCLC cells exhibiting pathway activation both in vitro and in vivo. Although NSCLC cells express Shh, they are not sensitive to cyclopamine and do not demonstrate cell autonomous pathway activation in vitro. However, NSCLC cell lines which signal to adjacent fibroblasts in vitro are growth inhibited by cyclopamine in vivo, suggesting that tumor-stromal interactions mediated by Shh promote malignant growth. These data show that activation of the Shh pathway promotes the malignant behavior of lung cancer, and that inhibition of this pathway may represent a novel mechanism-based therapy. Outside of studies in CNS tumors, this is the first direct demonstration of Hh pathway activation in any human cancer. Moreover, our studies show that this phenomenon is not a general feature of carcinomas, but is restricted to epithelial systems in which Hh signaling plays a role in development. We propose to establish inhibitors of Hh signaling as clinically useful therapies in lung cancer using an approach integrating human tumor tissue arrays, molecular and cell biology studies, mouse models and basic pharmacology. First, we will identify the prevalence of Shh pathway activation in lung cancer and pre-malignant airway tissue using immunohistochemical markers. Then, using genetically engineered reporter cell lines and mouse models, we will study the pharmacologic effect of Hh pathway inhibitors on tumor growth, pathway activation and tumor-stromal interactions. Using these preclinical models, we will then perform delivery, dosing and toxicity studies as a rational basis for eventual phase one studies in humans. This research plan will firmly establish the importance of Shh signaling in lung cancer, and provide a rational, mechanism based approach for the treatment of lung cancer with inhibitors of the Shh pathway.
Project 6 This project will target the enzyme, fatty acid synthase (FAS), for the treatment of lung cancer. Our preliminary studies have found that the vast majority of non-small cell lung cancers express high levels of this enzyme compared to normal tissues. This increased expression of FAS is signifcant because inhibition of this enzyme in cancer cells leads to a metabolic imbalance and cellular apoptosis. In a series of in vivo experiments, we found that treatment of orthotopic xenografts of human mesothelioma cells with an agent that inhibits FAS essentially abolished the growth of established tumors. Furthermore, in preliminary experiments, we found a promising anti-tumoral response of lung cancer orthotopic xenografts (in nude rats) treated with an FAS inhibitory compound. Importantly, these treatments did not result in any recognizable damage to normal tissue but did lead to dose-limiting anorexia. The proposed studies will further develop the use of FAS inhibitory therapy for lung cancer treatment. In the first phase of our preclinical studies, we will compare several novel F AS inhibitory agents, using in vitro and in vivo experimental systems, to identify a lead compound with high level of activity against lung cancer cells and tolerable levels of toxicity/ anorexia. In the second phase of the preclinical studies, we will optimize dosing protocols for the treatment of lung cancer orthotopic xenografts using this compound. This optimization of treatment protocols could be useful for designing treatment protocols to be applied in the clinical setting. The third pre-clinical aim of this project is to examine the effects of the F AS inhibitory compound when used in combination with other agents, such as those currently used to treat lung cancer or being evaluated for treatment of lung cancer. Because the F AS target represents a pathway distinct from those targeted by other compounds, there is a significant potential that such combinations could have synergistic antineoplastic activity, thus allowing reduction of doses of the respective agents. The final aim of this project is to initiate a phase I clinical trial for a compound identified by the preclinical studies to have the best potential for lung cancer treatment. Successful completion of this phase I trial and the preclinical modeling studies will provide a framework for further evaluation of an F AS inhibitory compound in the treatment of lung cancer.
Core 1 This core is responsible for enabling the PI to manage all the parameters of the SPORE. It is designed to accomplish this with low-cost, yet efficient administration and communication in order to divert as little funds as possible from research activities.
Core 2 The purpose of this shared resource is to provide human lung cancer tissues, other related biological specimens, correlative clinical and pathological data, and expert pathologic consultation for investigators of this Lung Cancer SPORE. The Human Tissue and Pathology Core has been in existence since 1992 under the support of the Lung Cancer SPORE. At the end of the year 2000, the frozen specimen inventory of the Core included 907 banked lung tumor resection specimens, 875 lymph node samples, 188 peripheral blood (plasma or serum) samples, 235 bronchial margin specimens, and 131 bronchiolalveolar lavage samples. In addition to these frozen specimen resources, the Core has collected paraffin-embedded samples of lung cancers and related pre-invasive lesions. In collaboration with the University of Colorado Lung SPORE, many of the paraffin embedded lung cancer samples have been assembled into tissue microarrays, which provides an important resource for many gene-expression studies. The Tissue and Pathology Core also collates and stores pathological, clinical, and outcome data for patients relevant to lung cancer specimens. Through an initiative of the Core, a relational database has been established to allow investigators ready access to this data. Finally, the Tissue and Pathology Core investigators are active consultants and collaborators for investigators of the SPORE and other lung cancer research scientists. For example, the SPORE has supported participation of our pathologists in inter-institutional efforts to standardize nomenclature for precursor lesions of lung cancer and of mouse models of lung cancer. The tissue collection efforts and collaborative participation of the Core investigators have contributed to 36 publications in peer reviewed literature during the past funding period and, in coming years, the Core will continue to provide a valuable resource to SPORE investigators and the lung cancer research community.
Core 3 The Biostatistics core of the proposed Johns Hopkins SPORE in lung cancer will consist of experienced members of the Division of Biostatistics in the Oncology Center, and support staff within the same division. A long history of collaboration already exists between the members of the core and several principal investigators, including the SPORE director. This core is designed to:
The Core will have an integral role in the scientific development, execution, and analysis of all projects in the SPORE. Core investigators have extensive and complementary experiences in quantitative methods for biomedical applications, including both clinical and basic science studies. They are committed to taking a direct interest in the substantive issues being investigated; to participating in regular project and program meetings, and to providing rigorous and innovative input on all quantitative matters arising in the projects. By contributing to multiple projects, they will also be in a position to promote interdisciplinary interactions among projects. Although funds are not requested to support it through this core, this resource will also provide any needed bioinformatics consultation to projects in the program, because a strong resource exists within the biostatistics program by virtue of its role in other SPORE programs. Such support includes designing new research databases and interfaces for data entry, data retrieval, patient or sample tracking, and procedures to ensure data quality, integrity, and confidentiality. Full analytic capabilities are also available.
Developmental Research Program The developmental funds for the Lung Cancer SPORE have been used to support two principle functions that have proven to be vital for the success of the SPORE. Initially, small pilot projects were emphasized which were solicited from Cancer Center and Johns Hopkins Medial Institutions faculty and selected by a committee of the entire major project PI's. This approach elicited some interesting science but we found that an additional approach could be imposed which yielded even greater benefit to the overall goals of the SPORE. From the original pilots, we selected two to perpetuate and nourish at a developmental level over the last funding period because either the translational potential of the work was increasing during the work and/or the involvement of the PI and inherent technology greatly enriched the entire culture of the SPORE. One of these projects has become a full project in the SPORE with studies that are at the highest level of translational research for this competitive renewal (Project 3). Another has developed a spin off with very high translational potential that will be continued at a developmental support level with high likelihood for becoming, or merging with, a major project. For the competitive renewal, we are now in a position to initiate new searches for pilot projects and several are already in hand for potential funding. These are included as examples from which the next selections for funding will be made. List of Investigators Stephen B. Baylin, M.D. Philip Beachy, Ph.D. Steven Belinsky, Ph.D. David Berman, M.D., Ph.D. Shyam Biswal, Ph.D. Julie Brahmer, M.D. Malcolm Brock, M.D. David Ettinger, M.D. Ed Gabrielson, M.D. James Herman, M.D. Timothy C. Kennedy, M.D. Francis Kuhajda, M.D. William Palmisano, Ph.D. Steven Piantadosi, M.D.-Ph.D. Martin Pomper, M.D. David Sidransky, M.D. Neil Watkins, Ph.D. William Westra, M.D. Stephen Yang, M.D. Rex Yung, M.D. |
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