|
|
||||
|
|
|
|
|
 |
 |
 |
 |
 |
 |
 |
 |
|
|
The Johns Hopkins University T.C. Wu, M. D., Ph.D. Overall Abstract Project 1: Markers of Progression to Cervical
Cancer in Rural India Cervical cancer is the number two leading cause of cancer death in women worldwide. HPV is the primary etiological agent of cervical cancer, and cervical cancer is therefore an entirely preventable disease. This project will focus on establishing an infrastructure for screening and diagnosis of women who have a high incidence of cervical cancer in a specific geographic region. The project will thus extend an international HPV research presence into an afflicted geographic region where the population may be able to benefit most from translational research and clinical applications. The proposed study site is Medchal Mandal, a rural community of about 40,000 individuals in India. This community possesses a fully equipped hospital sponsored by SHARE (Science Health Allied Research and Education), an American non-profit group that provides affordable and effective community-oriented health care and education to rural populations. This unique patient population and the well-equipped health care facilities and personnel in this community provide a unique resource to study cervical cancer. An extensive study will be performed to 1) compare four screening methods (Pap smear, Visual Inspection of the Cervix (VIA), HPV DNA in clinician-collected cervical swabs, and HPV DNA in self-collected vaginal swabs) for their ability to identify prevalent disease and to predict incident disease; 2) to characterize viral genotype, viral variants, viral persistence, viral load, and integration for their role in disease progression; 3) to evaluate cellular markers such as p16 overexpression, loss of FHIT expression, gain of chromosome 3q, and altered patterns of methylation for their role in disease progression; and 4) to correlate the viral and cellular markers of disease progression. The infrastructure in this rural Indian community will provide an opportunity to evaluate these markers in a community of women with a high prevalence of squamous intraepithelial lesions (SIL) and cervical cancer and allow for aggressive follow-up evaluations and care. Dr. Keerti Shah has designed the study and will oversee the characterization of molecular markers. Dr. Kathleen Cho will serve as a co-PI on this project, providing her expertise on molecular markers for cervical cancer progression, as well as reagents and data from Project 2. Drs. Shah and Cho have previously worked together on cervical cancer pathogenesis when Dr. Cho was on the faculty of Johns Hopkins from 1990 to 1998. Dr. Dorothy Rosenthal will advise on cytology and pathology procedures and help in the quality control of these test results. Implementation of this proposal will predictably reduce the prevalence and incidence of HPV and cervical cancer in this population.
Project 2: Identification of Molecular Markers for Cervical Cancer Progression This project seeks to identify molecular markers associated with, and perhaps responsible for progression of SIL to cervical cancer. Candidate genes will be tested for their ability to confer invasive potential to HPV-immortalized keratinocytes or cells derived from SIL. Using a large collection of cervical cancer tissue material as well as the technology of Affymetrix oligonucleotide microarrays, this project will be able to generate comprehensive data on gene expression profiles of tumor specimens. This project will 1) employ Affymetrix oligonucleotide microarrays to identify genes differentially expressed in HSIL (CIN3) versus invasive cervical carcinomas (in primary tissues and cell lines), 2) determine whether expression of selected candidate genes can serve as markers for identifying high-grade preinvasive lesions with increased likelihood of progression to invasive carcinoma, and 3) determine if selected candidate genes associated with the invasive phenotype confer invasive properties to HPV-immortalized keratinocytes or cells derived from human SIL. Dr. Kathleen Cho will coordinate the use of microarrays and the organization of microarray data for identifying candidate genes relevant to cervical cancer progression. Dr. Carolyn Johnston will oversee tissue collection and organization as well as eventual use of these markers in clinically relevant assays, prophylactics, and/or therapeutics. Dr. Cho will utilize tissues from Project 1 to validate her results with specimens from a different geographic area (India). Successful implementation of these aims would facilitate our understanding of cervical cancer pathogenesis and may elucidate potentially useful markers for prognosis/susceptibility to development of invasive cervical cancer as well as targets for developing immunotherapy. Project 3: Development of a Pan-Oncogenic HPV Preventive
Vaccine The long-term goal of this project is to eliminate HPV-related cancers through development of a single prophylactic vaccine effective against all oncogenic HPV types i.e. a pan-oncogenic HPV vaccine. Papillomavirus has only two capsid proteins, L1 and L2. Although immunodominant neutralizing epitopes are displayed on the major capsid protein, L1, the antibodies are highly type-specific. By contrast, in vitro neutralization studies with L2 antisera demonstrate cross-reactivity and suggest common epitopes in genital HPV L2 proteins. Importantly, vaccination with L2 protein protects animals from experimental infection with cutaneous and mucosal papillomaviruses. Since L2 is critical for papillomavirus infection and the existence of cross-neutralizing epitopes in L2 has been documented, the design of a simple pan-HPV prophylactic vaccine derived from L2 sequences is potentially promising. This study seeks to 1) determine whether protection from infection after vaccination with L2 is mediated by neutralizing antibody in a rabbit model (Neil Christensen, Ph.D., project co-investigator, will perform these studies at Penn State University, Hershey, PA), 2) identify cross-neutralizing epitopes in high-risk genital HPV L2 proteins and enhance their immunogenicity and cross-reactivity, 3) evaluate a clinically promising therapeutic HPV-16 L2-E6-E7 fusion protein vaccine (in collaboration with Cantab/Xenova Pharmaceuticals) for the generation of antibodies and their ability to neutralize a broad range of genital HPVs, and 4) investigate natural protection against HPV infection generated by antibody against L2 neutralizing epitopes by comparing natural acquisition of HPV-16 or other oncogenic types in patients with or without antibody specific for HPV-16 L2 neutralizing epitopes. The patient sera will be derived from the Guanacaste Project, the HIV Epidemiology Research Study (HERS) and Womens' Interagency HIV Study (WIHS) natural history studies, for which long-term follow-up is available. Dr. Christensen will determine whether antibodies affect protection after vaccination with L2. Dr. Roden will identify epitopes relevant to L2 and characterize neutralizing antibody generated by vaccination of patients with the L2 fusion protein vaccine. Dr. Viscidi will perform sero-epidemiologic studies and participate in the further clinical implementation of L2-specific assays and vaccines. The implementation of the aims of this project will contribute to the long-term goal of developing a single L2-specific prophylactic vaccine effective against multiple oncogenic HPV types.
Project 4: Human Immunological Responses to Chimeric L1/L2-E2-E7 VLP Since HPV infection does not produce viremia, antibody-mediated neutralization of the viral inoculum must occur in the genital tract. Our preclinical studies suggest that neutralizing antibody titers in vaginal lavage vary dramatically across the female reproductive cycle. Therefore, Project 4 will examine humoral responses of patients to the chimeric L1/L2-E2-E7 VLP vaccine in cervical secretions across the menstrual cycle. While vaccination with HPV virus-like particles (VLPs) comprising L1 only induces high titer serum neutralizing antibodies and protects animals from experimental papillomavirus infection, the efficacy of L1 VLP vaccination against venereal HPV transmission has not been tested. L1 VLPs do not generate therapeutic effects for established or breakthrough HPV infections that have escaped antibody-mediated neutralization. The control of these established HPV infections most likely requires therapeutic T cell-mediated immunity against other HPV antigens that, unlike L1, are expressed in infected basal epithelia, such as the viral early proteins E2 and E7. To increase the number of viral antigen targets for cell-mediated immune responses in a VLP-based vaccine, Dr. John Schiller (NCI) and Dr. Richard Roden have developed chimeric HPV VLPs consisting of the L1 major capsid protein plus the entire E2 and E7 nonstructural papillomavirus proteins fused to the L2 minor capsid protein. These chimeric VLPs retain the capsid morphology and ability to elicit high titers of neutralizing antibodies, while also inducing CD8-mediated E7-specific antitumor immunity in preclinical studies. Production of clinical grade HPV-16 L1/L2-E2-E7 chimeric VLP for phase I/II clinical trials is underway, and the Phase I/II clinical trials will be run at The Johns Hopkins University Center for Immunization Research by Dr Harro with Dr. John Schiller and Dr. Doug Lowy at NCI. Project 4 aims to examine humoral immune responses at the cervix, and to develop and utilize assays for the E7 and E2-specific cellular immunity generated by vaccination of patients with the chimeric L1/L2-E2-E7 VLP vaccine. This will allow for comparison with other vaccine strategies and correlation with clinical outcome. This project will take advantage of patients enrolled in phase I/II clinical trials of L1 and chimeric VLP at Johns Hopkins run by Dr. Harro. Dr. Harro will thus collect blood and cervical secretions from this patient group and Drs. Harro and Roden will examine humoral responses to vaccination with chimeric VLP vaccination in the serum and in the genital tract across the menstrual cycle. Dr. Roden, with the assistance of Dr. Pardoll and Dr. Schneck, will also design and implement immunological assays to characterize the HPV16 E2 and E7-specific cellular immune response of patients to chimeric VLP vaccination. Project 5: Vaccination with Sig/E7(detox)/HSP70 DNA to Treat
Patients with HPV-Associated High Grade Squamous Intraepithelial
Lesions with or without HIV The goal of this project is to determine the toxicity and dosage of an intracellular targeting strategy Sig/E7(detox)/HSP70 DNA in a clinical setting, to assess clinical and immunological responses to vaccination, and to compare vaccination outcome in HIV-seropositive and HIV-seronegative patients. Previously, we have developed a vaccine linking HPV-16 E7 antigen to heat shock protein 70 (HSP70), which enhances MHC class I presentation of E7 to CD8+ T cells, resulting in a potent E7-specific CD8-dependent, CD4-independent antitumor effect. This is particularly relevant to HIV-positive patients, who may develop low CD4+ T cell counts over time. These encouraging preclinical results have prompted us to consider applying this DNA vaccine to patients with high-grade squamous intraepithelial lesions who are HIV-negative or HIV-positive but asymptomatic with CD4 + T cell counts above 350. These vaccines incorporate a minimally mutated form of E7, termed, E7(detox), which has disrupted Rb-binding function of the E7 protein but preserves antigenicity. Our recent studies indicate that addition of signal peptide (Sig) to E7(detox)/HSP70 enhances E7-specific CD8+ T cell immune responses following intramuscular immunization, an effect comparable to that generated by gene gun immunization. We plan to: 1) determine the safety and toxicity of vaccinating HIV-positive and HIV-negative HSIL (CIN3) patients with Sig/E7(detox)/HSP70 DNA; 2) evaluate clinical responses to Sig/E7(detox)/HSP70 DNA vaccination; 3) characterize E7-specific humoral and T cell-mediated immune responses to Sig/E7(detox)/HSP70 DNA vaccination; and 4) characterize infiltrating immune cells and cytokine profiles, and correlate these data with the status of HPV and pathologic changes in the biopsy lesions before and after vaccination. Dr. Pardoll will oversee the design and implementation of immunological, clinical, virologic, and pathologic assays used in this project. Dr. Trimble will organize and oversee the design and execution of the clinical trial. Dr. Wu will design novel human immunological assays to characterize E7-specific immune responses in HIV-positive and HIV-negative patients. This project will allow us to evaluate the feasibility, safety, and immunogenicity of vaccination with Sig/E7(detox)/HSP70 in HSIL (CIN3) patients and correlate immunologic parameters with clinical outcomes. In addition, this project will allow us to compare vaccine effects in HIV-positive and HIV-negative patients and to test if Sig/E7(detox)/HSP70 DNA can lead to potent antigen-specific CD8+ T cell immune responses and control HSIL (CIN3) in patients with HIV despite their compromised CD4+ T cell immune responses.
Project 6: Combination of Antigen-Specific Cancer Immunotherapy and Anti-Angiogenesis to Treat Patients with Advanced Cervical Cancer Current therapies for advanced cervical cancer have minimal efficacy. However, antigen-specific cancer immunotherapy and anti-angiogenesis have emerged as two attractive strategies for cancer treatment. An innovative approach that combines both mechanisms will likely generate the most potent anti-tumor effect. We tested this approach using calreticulin (CRT), which has been shown to enhance MHC class I presentation of linked antigen and exhibit an anti-angiogenic effect. We linked CRT to HPV-16 E7 in a DNA vaccine and found that vaccination of mice with CRT/E7 DNA led to enhanced E7-specific CD8+ T cell immune responses and an anti-tumor effect against an E7-expressing tumor cell line, even in the absence of T cells. Additional assays confirmed the anti-angiogenic effect generated by CRT, even in nude mice, suggesting that this treatment may hold promise for patients that have received extensive prior chemotherapy. Thus, cancer therapy using CRT linked to a tumor antigen holds promise for effectively treating tumors by combining antigen-specific immunotherapy and anti-angiogenesis. More recently, we found that the CRT/E7 DNA vaccine demonstrated significant potency against established E7-expressing murine tumors with down-regulation of MHC class I molecules. Our encouraging findings have prompted us to test if vaccination with CRT/E7(detox) DNA can lead to a therapeutic effect against HPV-associated advanced cancers, since a significant proportion of advanced stage cervical carcinoma has been shown to exhibit down-regulation of MHC class I molecules. Therefore, in the current proposal, we plan to test if treatment of HPV-associated advanced cervical cancer patients with repeated CRT/E7(detox) vaccination is safe and capable of generating a therapeutic effect leading to reduction of viral load, infiltration of relevant immune cells, and improved clinical outcome. In addition, we will determine if repeated vaccination of CRT/E7(detox) DNA in patients with HPV-associated advanced cervical cancer can generate reduction of microvessel density in tumors. Dr. Wu will design, refine, and implement the immunological, clinical, virologic, and pathologic assays to be used in project 6. Dr. Armstrong will be responsible for clinical trial design, patient enrollment, and other clinically relevant activities.
Core A The Administration/Communication Core is proposed to facilitate coordination and oversight of all Program activities. This core will include a basic science director (Dr. T.-C. Wu) and a clinical research director (Dr. Frederick Montz). Dr. Wu will be responsible for coordinating basic scientific efforts and the coordination of individual projects. Dr. Montz will oversee patient identification, enrollment, and patient monitoring in the context of the Core. This resource will also fund a Clinical Research Coordinator who will interact with the Tissue Core and the 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 monitoring of patient safety, adverse events, and data management and confidentiality will be provided by the data safety monitoring board, as well as the Clinical Research Management Group. 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.
Core B The Biostatistics and Bioinformatics Core of the proposed Johns Hopkins SPORE in cervical cancer will consist of Dr. Mei-Cheng Wang, Dr. Rafael Irizzari and a programmer in the department of Biostatistics, and Dr. Elizabeth Garrett, a member of the Division of Biostatistics in the Oncology Biostatistics Center. The investigators have extensive experience in applying conventional and up-to-date statistical techniques to the analysis of both clinical and laboratory data in cancer and AIDS studies. The Core will have an essential and integral role in scientific development, execution, and analysis of all projects in the SPORE for cervical cancer. 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, participating in regular project and program meetings, and providing rigorous and innovative input on all quantitative matters arising in the projects. The Core is designed to:
Core C Organized collection and expert pathologic evaluation of human tissues and biologic fluids is pivotal to the translational mission of this SPORE project. Thus this Tissue/Pathology Core is designed to provide well-characterized human biological specimens to researchers participating in the Johns Hopkins Cervical Cancer SPORE and other similar research efforts. In addition to the growing need for sophisticated sample acquisition, investigators increasingly depend on Pathology support to ensure proper tissue preparation and characterization for selected studies. This Core will also provide expert pathologic evaluation of specimens and technical support. Specifically, this core will:
Developmental Research Program The Developmental Research Program is an important component of the SPORE and critical to the long-term fight against cervical cancer. It provides an avenue for soliciting new research ideas and for developing innovative high-risk, but high-impact projects to stimulate cervical cancer research in the context of the SPORE. Pilot studies provide investigators with the resources to conduct translational research consistent with the SPORE's objectives. This program will encourage participation from a broad range of investigators at Johns Hopkins by providing support for pilot projects with the potential to develop into more fully developed translational projects. It will also encourage and facilitate the development of new research directions, methodologies, and collaborations. Career Development Program The Career Development Program will allow the Cervical Cancer SPORE to attract and stimulate young investigators to carry out translational research related to cervical cancer. Candidates will submit an application and be evaluated through a careful selection process involving a career development committee headed by Dr. T.-C. Wu as well as the SPORE steering committee. Dr. Robert J Kurman will assist the translational and clinical efforts of awarded investigators. Recipients of the award will be reviewed annually and investigators are required to submit an annual progress report subject to the aforementioned review process. List of Investigators Debbie Armstrong, M.D. Robert Bristow, M.D.
Kathleen Cho, M.D. Clayton Harro, M.D. Carolyn Johnston, M.D Robert Kurman, M.D. Drew Pardoll, M.D., Ph.D Richard Roden, Ph.D Keerti Shah, M.D., Ph.D Cornelia Trimble, M.D. Raphael Viscidi, M.D. Mei-Cheng Wang, Ph.D T.-C. Wu, M.D., Ph.D |
 |
|||
|
|