Cervical Cancer SPORE Program Description

 Introduction

Approximately 500,000 women worldwide develop cervical cancer each year and it is the second leading cause of cancer death in women. Despite well-established cervical cancer screening programs, the incidence of invasive cervical cancer in the US has been increasing at a rate of 3% per year since 1986 (SEER Cancer Statistics Review).  Thus, cervical cancer continues to be a major health care problem in the U.S. and worldwide.  The convergence of advances in our understanding of the pathogenesis of cervical cancer and immunotherapy over the last 10 years provides a timely opportunity for investigators from our institution to apply for a SPORE grant at this time.   While progress in understanding the etiology of other major epithelial cancers has been relatively slow, epidemiologic/virologic data over the past 15 years have demonstrated that oncogenic human papillomaviruses (HPVs) are the primary causal agent of cervical cancer. Evidence linking HPVs to anogenital cancer comes from a wide variety of clinical, pathologic, epidemiologic and laboratory studies.  More than 99% of cervical cancers and over 90% of their precursor lesions, squamous intraepithelial lesions (SIL), contain HPV DNA (Walboomers, J Pathol, 1999).  In addition, it has been demonstrated that the expression of HPV oncogenes is necessary for maintenance of the malignant phenotype.  Thus, it has become widely recognized by investigators at a number of academic institutions and pharmaceutical companies that preventive and therapeutic strategies aimed at HPV are the most effective way of treating and potentially eradicating cervical cancer.  Moreover, recent advances in molecular immunology and in understanding the molecular pathogenesis of HPV and cervical neoplasia will permit clinical implementation of these approaches within the 5 years of this SPORE program.  A significant amount of the work in this area has been done in the past decade by investigators at Johns Hopkins.  These investigators with expertise in molecular and cellular biology, virology, immunology, pathology, medical and surgical oncology, and epidemiology of cervical cancer continue to work together with the primary long-term goal of translating scientific advances made in the laboratory into novel preventive and therapeutic strategies to reduce cervical cancer incidence, morbidity, and mortality.

The SPORE program incorporates translational research in 3 major program areas – Program 1: Molecular Markers Relevant to the Screening, Diagnosis, and Prognosis of Cervical Cancer (Projects 1 and 2), Program 2: Preventive Vaccines for Cervical Cancer (Projects 3 and 4), and Program 3: Innovative Therapeutic Vaccines to Control HPV-Associated Precursor Lesions (Project 5) and Advanced Cervical Cancer (Project 6).

The goal of Program 1 (Projects 1 and 2) is to identify and evaluate molecular markers relevant to the screening, diagnosis, and prognosis of cervical cancer.  Project 1 will implement population-based screening and diagnosis and evaluate biological markers of disease progression in a target population of women in India who have a high incidence of cervical cancer.  The goal of Project 2 is to identify and characterize molecular markers that are relevant to tumor progression.  These markers are the key to identifying the subset of HPV-infected patients likely to progress to cervical cancer.  Such markers would not only contribute to our understanding of cervical cancer pathogenesis, but also would be useful for prognosis and as therapeutic targets.  The goal of Program 2 (Projects 3 and 4) is to reduce the incidence of cervical cancer by developing vaccines that effectively prevent HPV infection.  For cancers in which an infectious agent has been identified, vaccination against these agents has proven to be the simplest and most effective approach for prevention and treatment.  The goal of Program 3 (Projects 5 and 6) is to reduce morbidity and mortality associated with cervical cancers and their precursor lesions using innovative vaccines to treat patients with established disease.

While oncogenic HPV is generally able to evade the immune system, recent advances in our understanding of antigen presentation and cellular immunity have led to novel vaccination strategies to overcome these difficulties.  We have included four vaccine projects in this SPORE program because it has become eminently clear that successful treatment and prevention, and indeed eradication, of cervical cancer is not merely a pipe dream but a reality based on the elimination of HPV infection.

The goals of this SPORE application are consistent with the high-impact priorities of the NCI Gynecologic Cancers Progress Review Group (as described in November 2001): 1) Identify precursor lesions, markers of risk and early detection, molecular disease classifications, prognostic indicators, and new targets for prevention and treatment; (2) Develop effective human papillomavirus (HPV) vaccines to prevent biotransmission and development of neoplasia; and (3) Conduct research to (a) understand and improve the quality of life of gynecologic cancer patients; and to (b) reduce or eliminate disparities related to care among patients with gynecologic cancers (see attached Priorities of the NCI Gynecologic Cancers Progress Review Group in appendix 1).  Importantly, major headway towards achieving our goals and the high-impact priorities of the NCI can be accomplished within 5 years.

The diversity of our translational research objectives is reflected by the focus of each project on different targets throughout the disease process.  Chart 1 illustrates the scope of each project with regard to the course of HPV infection and cervical carcinogenesis.

Chart 1: Targeting HPV Infection and Cervical Carcinogenesis

This SPORE Project is a highly coordinated and collaborative interdisciplinary program consisting of basic and clinical research with the overall goal of elucidating the molecular pathogenesis of cervical cancer and the development of vaccines that will treat and prevent this cancer.  The organization of the SPORE program joins the efforts of Pathology, Oncology, Molecular Microbiology and Immunology, Gynecology and Obstetrics, Pediatrics, Medicine, Public Health, the Cervix Center, and the Cancer Center at Johns Hopkins and the departments of Pathology, Obstetrics and Gynecology, and Oncology at the University of Michigan, providing support and cohesion for the related projects through integrated Core resources.  The program also builds on prior RO1 and PO1 funding and the infrastructure developed within the departments that are involved.  As a result, the work of each researcher will be enhanced and accelerated by the SPORE.  The program will improve the clinical care of patients with cervical cancer to a much greater degree than the efforts of the individual researchers working separately.

Description of Individual Projects

 Project 1: Markers of Progression to Cervical Cancer in Rural India, led by Keerti Shah, M.D., Dr.PH., and Kathleen Cho, M.D.

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, led by Kathleen Cho, M.D. and Carolyn Johnston, M.D.

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, led by Richard Roden, Ph.D., Raphael Viscidi, M.D. and Neil D. Christensen, Ph.D.

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, led by Clayton Harro, M.D., Sc.M., Richard Roden, Ph.D., Jonathan Schneck, M.D., Ph.D and Drew Pardoll, M.D., Ph.D.

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, led by Drew Pardoll, M.D., Ph.D., Cornelia Trimble, M.D., and T.-C. Wu, M.D., Ph.D.

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, led by T.-C. Wu, M.D., Ph.D. and Deborah Armstrong, M.D.

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.

Description of Individual Cores

These six projects are supported by three Cores.

Core A is the Administration and Communication Core led by T.-C. Wu, M.D., Ph.D. This core is responsible for administration of the SPORE and its duties include coordination, evaluation, and planning of research directions; budgetary oversight; review of progress and safety; and communication within the SPORE and with the institution, other committees, NCI program staff, and other SPORE investigators at Johns Hopkins and at other institutions.  The clinical research director is responsible for overseeing patient identification, enrollment, and patient monitoring and interacting with the Data Safety Monitoring Board.

Core B is the Biostatistics and Bioinformatics Core led by Mei-Cheng Wang, Ph.D., assisted by Elizabeth Garrett, Ph.D. and Rafael A. Irizarry, Ph.D.  This core is responsible for overseeing issues related to study design, data management, and data analysis and will serve as an important component of translational research activities.

Core C is the Tissue and Pathology Core coordinated by Robert Kurman, M.D., assisted by Robert Bristow, M.D. and Brigitte Ronnett, M.D.  This core is responsible for collection, organization, and distribution of laboratory and clinical specimens, including those samples generated during the proposed clinical trials, to SPORE investigators.