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The Johns Hopkins University School of Medicine SPORE in Head and Neck Cancer David Sidransky, M.D. Purpose and Intent: The goal of this SPORE Project is to reduce the morbidity and mortality of head and neck cancer through a highly coordinated program consisting of basic and clinical research focusing on the molecular parameters associated with the development and progression of this deadly disease. The organization of the program joins the efforts in Otolaryngology, Oncology, Public Health, and the Cancer Center at Johns Hopkins in fighting this entity. The SPORE Program itself will join researchers from clinical and basic science disciplines and provide support and cohesiveness for the related projects through integrated Core resources. This program also builds on prior RO1 funding and specific infrastructure developed within the Division of Head and Neck Cancer Research and as a result, the work of each researcher will be enhanced, expanded and accelerated. The effect of the program and the clinical care of patients with head and neck cancer will be greater than the sum of the efforts of the individual researchers working separately. The projects in the SPORE program are united around the common research goals of generating and applying information about the molecular basis of HNC. Through the use of high throughout technology we will test for genetic alterations and tumors and paired clinical samples and bring this information into the clinical arena to better understand the causes of the disease, the possible benefits to the patients, and identify new targets for therapy. Through these goals, we hope to develop new strategies for early detection, prognostication, treatment selection, and monitoring of head and neck and thyroid tumors through improved understanding of the progression of disease and the application of that knowledge into the clinical arena. Several avenues of inquiry are incorporated into the proposal which are broadly separated into diagnostic and therapeutic approaches. The diagnostic approaches center around the use of new methylation and microsatellite alteration tests in the paired serum and saliva of patients with head and neck tumors. In addition, we make use of community programs that identify patients at high risk for head and neck cancer and provide patient samples at risk for this disease. This will allow us to create a multi-varient model to determine which risk factors and molecular alterations predict the presence of premalignant lesions in which are likely to lead to tumor progression. These markers will also be used in the therapeutic approaches described below to help monitor patients and develop sensitive techniques to monitor tumor burden. Our therapeutic approaches will be based on identification of a subset patients have activated a specific tyrosine kinase. In SCC patients that are HPV positive, we will develop a novel E7/HSP70 DNA vaccination program for high-risk individuals. Patients will be treated and characterized for E7 specific immuno response and monitored with molecular markers. Patients that have activated EGFR will be treated with OSI-774 a novel tyrosine kinase inhibitor and molecular responses will be correlated with specific pathway activation. Preclinical models based on these leads will be used to identify additional targets and increase anti-tumor activity, resulting in a Phase I/II trial that may incorporate downstream inhibitors of the EGFR pathway. Patients with activated RET in thyroid cancer will also be treated with a novel TKI inhibitor and similarly monitored for a response. Thus, these strategies represent a cohesive interaction between diagnostic and therapeutic approaches that will benefit patients as never before. The SPORE program has the ability to achieve this goal because it optimally applies existing investigator talent, achievement and interest and uses the most promising current research directions in head and neck cancer.
PROJECT #1 The primary objective of this project is to field test a molecular screening assay developed in our laboratory for the early detection of malignant and premalignant disease of the upper aerodigestive tract. A secondary goal is to identify molecular markers indicative of a high risk of tumor progression for premalignant lesions. These goals will be approached using three specific aims. In aim one, the molecular test using microsatellite alterations and tumor-specific hypermethylation to detect neoplastic cells exfoliated from the mucosa of at-risk individuals participating in community based cancer screening programs will be applied and evaluated. New molecular markers developed in Project 2 will be added to a panel of markers already validated in pilot studies involving individuals with known head and neck invasive cancer. The rate at which these alterations are present in the at-risk but presumably healthy population in the screening sessions will be determined together with the association of genetic alteration with exposure to known risk factors for head and neck cancer (HNSC). These healthy subjects will then be followed for 2 years and then re-examined. Specific aim #2 seeks to identify molecular markers for progression that will be combined together with physical findings and exposure history into a multivariate predictive model to investigate the independence of the molecular factors as predictors of disease progression. Individuals who are identified at screening sessions or in a consortium of medical and dental clinics to have visible oral premalignant or benign mucosal lesions will be eligible for participation in Specific aim #3 which will investigate molecular markers in the premalignant lesions and exfoliated cells again looking for markers associated with progression to invasive cancer. Molecular markers will be compared with histologic grade of the index lesion as well as risk-exposure history in multivariate analysis to identify the most useful predictors of progression. All molecular markers will be analysed in extracted DNA taken from tissue biopsy, oral rinses and swabs, and peripheral blood plasma and compared with control DNA from peripheral blood leukocytes. Extracted DNA will be amplified using primers specific for microsatellites known to be frequently altered in HNSC and samples then separated on gels in order to identify tumor-specific alteration. Methylation markers will be assayed using Taqman fluorescence methodology. These studies promise to improve and validate an early detection paradigm that can then be applied in mass screening programs. The early detection of individuals likely to harbor HNSC through community screening programs would permit the diagnosis and treatment of the disease while it is still highly curable using current treatment methods. Accurate identification of individuals most likely to demonstrate progressive disease after presentation with premalignant lesions will permit the rational utilization of current and new treatment and preventive measures.
Project #2 We and others have demonstrated that silencing of tumor suppressor genes associated with promoter hypermethylation is a common feature of many human cancers. Loss of p16 activity through hypermethylation of the promoter is associated with loss of the expression and function early in the progression of head and neck cancer. Methylation of p16 and other selected methylated markers have been identified in the saliva and serum of patients with head and neck cancer. We propose to thoroughly catalog hypermethylated genes in squamous cell carcinoma of the head neck (HNSC) and will identify novel hypermethylated genes by: (1). A candidate gene approach based on testing genes found to be hypermethylated in other tumors or on the functional structure and biologic plausibility of the candidate gene and (2) A functional screen looking for genes that are up-regulated following treatment with demethylating agents (5-Aza-deoxycytidine). Newly identified hypermethylated genes will be characterized in tumor progression. Moreover, these genes will be tested as markers in cancer detection. Optimization of sensitive real-time PCR assays able to detect specific methylation will allow us to test these markers in paired saliva and serum samples from patients with head and neck cancer for initial analytical validation. A screening study in the larger SPORE proposal (project #1) will allow us to further test these markers and determine the feasibility of this approach for the early detection and monitoring of patients with head and neck cancer.
Project #3 Strong evidence of an etiologic association between HPV and a distinct subset of oropharyngeal squamous cell carcinomas, distinguished by the presence of high-risk HPV DNA in tumor cell nuclei, has recently been reported. Because viral oncoproteins E6 and E7 are consistently expressed in HPV-associated cancers and are tumor-specific antigens, patients with HPV-associated head and neck squamous cell carcinoma (HPV-HNSCC) may benefit from therapeutic strategies designed to augment the cellular immune response to HPV oncoproteins. In preclinical model systems, linkage of Mycobacterium tuberculosis heat shock protein 70 (HSP70) to the HPV 16 E7 antigen was demonstrated to significantly enhanced the potency of a naked DNA vaccine. Vaccination of mice with E7/HSP70 DNA increased the frequency of HPV 16 E7-specific CD8+ T cells by at least 30-fold compared to vaccination with wild-type E7 DNA and generated a significant antitumor effect against an E7-expressing tumor. This vaccine demonstrated significant potency against established E7-expressing murine tumors with down-regulation of Major Histocompatibility Complex (MHC) class I molecules, an important finding given a significant proportion of advanced stage HNSCC exhibit down-regulation of MHC class I molecules. Furthermore, repeated DNA vaccinations elicited qualitatively different cytotoxic T lymphocytes (CTL) with higher avidity and improved protective anti-tumor effects. An E7 gene with mutations in the critical Rb binding residues (termed E7 detox) was generated which failed to bind Rb, had no transforming activity, and yet maintained potent immunogenicity. Support from the NIH Rapid Access to Intervention Development program has been obtained to generate clinical grade E7(detox)/HSP70 in the pNGVL4a DNA backbone for clinical trials. Sequential phase I and II clinical trials of repeated intramuscular injections of the E7(detox)HSP70 DNA vaccine are proposed in patients with HPV 16-positive oropharyngeal squamous cell carcinoma. The phase I trial is designed to evaluate the feasibility and safety of adjuvant therapy with escalating doses of E7(detox)HSP70 DNA vaccination as well as the effect of dose on the generation of HPV 16 E7-specific CD8+ T-cells in peripheral blood. The phase II trial is designed to evaluate whether neoadjuvant therapy with repeated DNA vaccination alters the number, avidity and E7-specific cytotoxic activity of HPV 16 E7-specific CD8+ T-cells and whether successful generation of these cells results in infiltration of tumor by CTL and other immune effectors. In an exploratory analysis, subjects in the phase II trial will also be observed for evidence of clinical response to E7(detox)/HSP70 DNA by measuring the size of target lesions before and after vaccination and reporting time to disease progression and two-year progression free survival.
Project #4 The prognosis for patients with locally advanced head and neck squamous cell cancer(HNSCC) is generally poor despite some gains using the current standard of concurrent radiation(RT) and chemotherapy. The epidermal growth factor receptor/tyrosine kinase (EGFR/TK) signal transduction pathway is important in the regulation of multiple oncologic processes including angiogenesis, MMP secretion. Overexpression of EGFR and its ligands occurs in 80-90% of HNSCC and is associated with poor survival and resistance to RT. Blockade of EGFR augments the cytotoxicity of cisplatin and enhances radiation sensitization. We propose to study the small molecule selective EGFR/TK inhibitor OSI-774 with the goal of developing a regimen that combines OSI with RT/cisplatin and inhibitors of signaling proteins downstream of EGFR to optimize clinical effect. Our hypothesis is that it will be necessary to inhibit multiple pathways to totally eradicate tumor and prevent recurrence. We will build on our experience with a phase I trial of OSI-774 that established dosing and pharmacokinetic/ pharmacodynamic parameters (Hidalgo et al). We will conduct a phase I trial of OSI-774 + RT and + RT/cisplatin. In paired biopsies we will determine the effect of OSI on a broad spectrum of genes using cDNA arrays and effect on well-defined EGFR-related functions such as signaling, angiogenesis, apoptosis, cell cycle regulation. This will allow us to select other relevant targets which we will then evaluate in preclinical in vitro and in vivo models. We will test adding inhibitors of these downstream signaling proteins to our back-bone regimen of OSI-774 + RT/cisplatin performing the same molecular assessments as in the preceding clinical trial. In a third phase of this project we take our results from the laboratory back to the clinic and design a phase I/II trial based on the optimal activity seen in our preclinical model. This may be a fixed combination of OSI + downstream inhibitor or a variable combination based on pattern of gene expression and signal transduction activation in individual patients. Candidate molecules to be combined include inhibitors of angiogenesis, MMPIs, Cox-2 agents and MAPK or P13K inhibitors.
Project #5 Activation of the RET tyrosine kinase is involved in approximately 50% of cases of medullary thyroid carcinoma (MTC) and 20-25% of cases of papillary thyroid carcinoma (PTC). For medullary thyroid carcinoma, there is no effective therapy other than surgery. In this proposal, we will develop therapeutic approaches for MTC and PTC, based on inhibition of RET. We have shown that the indolecarbazole compunds of CEP-701 and CEP-751 inhibit RET and induce apoptosis in MTC cells in culture. Focusing mainly on MTC, this proposal will develop this observation, toward effective therapy for these diseases in humans. The effect of CEP-701 and CEP-751 on inhibition of R ET activity and MTC growth will be evaluated in an animal model of human MTC. Combination therapy, using CEP-701 or DEP-751 and a conventional cytotoxic agent, will be examined. A library of tyrosine kinase inhibitors, structurally similar to CEP-701 and CEP-751, will be screened for more effective RET inhibitors. Correlative markers will be identified for RET inhibition, based on downstream effectors of RET signal transduction pathways. Immunological assays for these markers will be developed for future use in assessing the efficacy of RET inhibition in clinical settings. The effect of RET inhibition by CEP-701, CEP-751, or other compounds on several forms of RET commonly activated in MTC and PTC will be examined. In addition, the effect of RET inhibition therapy will be examined in tumors arising in the natural thyroid setting, in transgenic models of MTC and PTC.
ADMIN Core An Administrative Clinical Core is proposed to facilitate coordination and oversight of all Program activities by Dr. Sidransky. This core will include a basic science director (Dr. Sidransky) and a clinical director (Dr. Forastiere). Dr Sidransky will be responsible for coordinating basic scientific efforts and the coordination of individual projects. Dr Forastiere will coordinate patient identification, enrollment, and patient monitoring in the context of the Administrative Core. This resource will also fund a Clinical Director, and a Clinical Research Coordinator who will interact with the Tissue Core and a Biostatistics Core, as well as with personnel from each individual project to ensure that all patient information, specimens and results are properly collected and recorded in the computerized database. Appropriate data safety monitoring will be provided and clinical oversight will be provided by reporting to a data safety monitoring board, an internal advisory panel, and an external advisory panel. The Administrative Core includes key administrative personnel and will also coordinate essential Program interactions including all planning and evaluation activities, arranging and publicizing SPORE activities, coordinating advisory committee meetings, producing annual reports and performing analysis of budgetary matters.
Biostatistics and Bioinformatics Core The Biostatistics and Bioinformatics Core of the proposed Johns Hopkins SPORE in head and neck cancer will consist of three experienced members of the Division of Biostatistics in the Oncology Center, and database programmers from the Clinical Research Office within the same division. A long history of collaboration already exists between the Director of the Core and several Principal Investigators, including the SPORE Director. Teh 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 complimentary 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.
Tissue Core Over the past decade, knowledge about the pathogenesis of human tumors has been attributed to and limited by the availability of well-characterized human tissues. With this in mind, tissue/biologic fluid facilities have emerged as a means of overseeing tissue distribution for investigative studies. The purpose of the Shared Resource is to provide human tissues, biologic fluids, and expert pathologic interpretation for investigators on all of the projects. The Resource will collect, store, process, and distribute tissues and biologic fluids in a highly coordinated fashion. The Resource will collect tissue/biologic fluids in a manner that meets the needs of the individual investigators without compromising clinical patient care. The Resource will store these samples in such a way as to ensure long-term security and easy accessibility. The Resource will process samples so that they are suitable for further analysis. Finally, the Resource will distribute samples to investigators in a timely fashion. Specimens are collected under the supervision of pathologists with expertise in neoplasia of the upper aerodigestive tract in close collaboration with clinical specialists in this area and in similarly close collaboration with basic research investigators to maximize translational impact of the projects. A concerted effort to collect and bank tissue specimens and biologic fluids from the upper respiratory tract has been an ongoing effort since 1990. As of 9/1/01, the resource of freshly collected human biological specimens (not inclusive of the paraffin tissue bank) includes 1,060 banked head and neck carcinoma resection specimens, 10 pre-invasive mucosal dysplasias, 327 oral rinses/oral swabs obtained from at-risk but cancer-free individuals, and 2,728 histologically negative specimens including margin samples from cancer resection specimens, and 200 thyroid neoplasms. These materials have been collected with appropriate IRB approval and patient consent. The processing of these tissues to ensure suitability for investigative purposes has become increasingly refined. At the present time, the Shared Resource has ready access to a laser capture microdissection facility, a tissue array facility, and an immunohistochemistry facility. Distribution of these materials to various investigators including many of those participating in the SPORE has resulted in over 200 publications regarding carcinoma of the head and neck.
Developmental Research Program One critical aspect of the SPORE program is the ability to take novel diagnostic and therapeutic approaches and develop them during the SPORE funding. We have already identified smaller pilot projects that are not ready for full expansion in the translational setting. However, these individual pilots hold great promise and the SPORE program provides the flexibility for diminishing or increasing support for these as needed to ensure the development of the best translational approaches. These pilots include the development of a HPV detection assay in saliva and serum similar to those developed from microsatellite and methylation changes in SCC for HPV positive tumors. Identification of the strengths and barriers of molecular detection programs will also be assessed and garnished information will likely impact the use of these tests in the clinical setting. Methylation markers will also be extended to thyroid cancer detection in addition to our work in squamous cell carcinoma. Finally, molecular correlates of onyx 15 gene therapy in advanced head and neck cancers with altered p53 function will also be investigated. This promising approach will also benefit from molecular detection tests to assess tumor burden. Pilot studies have been selected through a competitive program over the past year at Johns Hopkins. These studies initially funded through the SPORE program will be constantly monitored and assessed by the steering group composed of all the PI's as well as the internal and external advisors. As these projects mature they will be terminated if they do appear to have a translational benefit or they will be expanded to benefit the patients that they can best benefit. As noted below, these pilot projects also provide great enthusiasm for career development for some of our most outstanding young researchers in the field.
Career Development Program The SPORE Program realizes that career development is an integral part of SPORE related activities. The Principal Investigator, Dr. David Sidransky, was initially funded through the Lung SPORE Grant a decade ago, which in no small part led to the launching of his career. Thus, Dr. Sidransky and a panel of senior investigators who understood career development have selected the most promising and outstanding researchers at Johns Hopkins for this effort in the SPORE program. These leaders of tomorrow have helped developed the major pilot projects as described above and will be closely monitored during the SPORE program. The steering committee (see below) with the help of the internal and external advisors will make sure these young investigators not only develop individually as scientists and clinicians but also develop into translational researchers. The success of the SPORE will thus not only be measured by scientific and clinical achievements but also by academic success for these young investigators. |
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