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FOX CHASE CANCER CENTER
OVERALL ABSTRACT Ovarian cancer is the number one gynecologic killer in the United States. New opportunities for diagnosis, prevention, and treatment will be dependent upon an expanded translational research effort. Fox Chase Cancer Center's Specialized Program of Research Excellence (SPORE) in Ovarian Cancer renewal application consists of five translational research projects, a Developmental Research Program, a Career Development Program, and five specialized Cores to support the research programs. These research projects represent a diversity of translational research objectives, including diagnosis, prevention, and treatment: 1) Mechanisms of COX-2 Inhibition in Ovarian Cancer Prevention; 2) AKT as a Biomarker of Ovarian Cancer Progression and a Target for Therapeutic Intervention; 3) Anti-Mullerian Inhibiting Substance Type II Receptor (MISIIR) Immunoconjugates to Detect and Treat Ovarian Cancer; 4) Molecular-tageted Therapy in Ovarian Cancer; 5) Immunotherapy of Ovarian Cancer: Taking Down the Barriers. The last Project is in collaboration with the Abramson Cancer Center of the University of Pennsylvania. A Biological Scientist and an Applied Scientist are Co-Principal Investigators on each Project. The specialized Cores include an Administrative Core, a Biosample and Tissue Procurement Core, an Ovarian Cancer Consortium for Research and Surveillance Core, a Mouse Engineering Core, and a Biostatistics and Data Management Core. Fox Chase Cancer Center has a long-term commitment to research in ovarian cancer and will provide additional institutional resources to support the goals of the SPORE application and will collaborate with other Ovarian SPORE institutions. Senior leadership is directly involved in the SPORE application. The Principal Investigator is the Senior Vice President for Medical Science. The Co-PI is the Program Leader of the CCSG Ovarian Cancer Program, and the President of Fox Chase Cancer Center serves on the Executive Committee. The Ovarian SPORE Program is a multidisciplinary collaboration of laboratory researchers and clinicians focused on decreasing morbidity and mortality from this disease. Based on progress in the initial funding period, the Fox Chase Cancer Center Ovarian SPORE Program is well positioned to accomplish this goal by prevention strategies based on the understanding of ovarian oncogenesis coupled with novel scientifically-based therapeutic approaches.
PROJECT 1 Epidemiological and experimental data support the use of non-steroidal anti-inflammatory drugs (NSAIDs), including specific inhibitors of Cox-2, as chemopreventive agents in a number of epithelial cancers. In general, it has been suggested that the inhibitors limit Cox-2-catalyzed production of prostaglandins, which may affect cell proliferation, apoptosis, anti-inflammatory responses, and angiogenesis. Based on our recent observations in ovarian cancers and from studies in animal models, we propose here a new mechanism for the chemopreventive activity of Cox-2 inhibitors in ovarian cancers, related to the integrity of the epithelial basement membrane. In the normal premenopausal ovary, the gonadotropins induce Cox-2 expression following the pre-ovulatory phase of follicular maturation. Cox-2 induction signals the initiation of the ovulatory phase, and inflammatory-like biological process. Moreover, prostaglandins, the products of Cox-2 activation, are believed to activate/induce collagenase and proteolysis and decrease the synthesis of the basement membrane components in both granulosa and ovarian surface epithelial cells. A recent study of pre-neoplastic lesions of human ovarian tumors suggests that the collagen IV- and laminin-containing basement membrane of the ovarian surface epithelium is lost prior to morphological transformation of the epithelial cells, suggesting that without an intact basement membrane, the surface epithelium represents a precursor lesion and subsequent genetic and epigenetic changes will lead to overt neoplastic transformation and tumorigenicity. By inhibiting Cox-2, the loss of basement membrane of the ovarian surface epithelium may be lessened and neoplastic transformation of the ovarian surface epithelial cells may be prevented. We propose to investigate the occurrence of basement membrane loss and Cox-2 overexpression in pre-neoplastic lesions of human ovaries from prophylactic oophorectomies of women from high-risk breast and ovarian cancer families. To investigate the mechanisms, we will also examine the effects and cellular signaling pathways of gonadotropin stimulation on the expression of Cox-2, collagen IV, laminin, and MMPs in ovarian surface epithelial cells in culture. The effect of Cox-2 overexpression will be assessed using ovarian surface epithelial-specific transgenic mice. We will also initiate a clinical trial to examine the effect of daily oral intake of Cox-2 inhibitors on the basement membrane integrity and occurrence of pre-neoplastic lesions in ovaries from prophylactic oophorectomies of women from high-risk breast and ovarian cancer families. The reduced loss of basement membrane are reduced number of pre-neoplastic lesions will be used as surrogate endpoints for the preventive activity of Cox-2 inhibitorsin ovarian cancer. These studies will help to understand the etiology of ovarian cancer related to gonadotropin stimulation and provide a mechanism(s) for the chemopreventive activity of Cox-2 inhibitors. The ultimate goal will explore the use of Cox-2 inhibitors for chemoprevention of ovarian cancer.
PROJECT 2 Activation of AKT promotes tumor cell survival, proliferation and invasiveness, suggesting that AKT may play a central role in tumorigenesis and therapeutic response. AKT is frequently activated in ovarian cancer and may occur early in tumor progression. Rapamycin targets AKT signaling via inhibition of mTOR, leading to G1 arrest in tumor cells with activated AKT. Fatty acid synthase (FAS) is often over-expressed in ovarian carcinomas, and its expression is regulated in part by AKT signaling. Enzymatic inhibition of FAS by specific pharmacologic agents induces apoptosis in tumor cells. Collectively, these data suggest that mTOR and FAS are promising targets for the treatment of ovarian cancer patients. The long-term objective of this proposal is to determine if mTOR or FAS inhibition by specific pharmacologic agents can be therapeutically efficacious in ovarian carcinomas, and whether response is dependent on the AKT status of a given tumor. The specific aims are: 1) Determine whether AKT activation and genomic imbalances occur early in the progression of human ovarian cancer. Immunostaining will be performed on putative preneoplastic ovarian lesions to determine if activation of AKT is an early event in ovarian tumorigenesis. To place AKT activation in the context of a model of ovarian tumor progression, array-CGH, an invaluable new tool that permits high-resolution analysis of genomic imbalances, will also be used to identify early somatic genetic changes in these same specimens. 2) Identify potential synergy between FAS inhibition or mTOR inhibition and various chemotherapeutic agents in ovarian cancer cell lines with or without constitutive activation of AKT. 3) Determine whether pharmacologic inhibition of AKT pathways can repress ovarian tumor formation. As a chemotherapeutic strategy, xenograft models of human ovarian cancer will be used to assay anti-tumorigenic effects of mTOR and FAS inhibitors. As a chemoprevention strategy, a transgenic ovarian cancer model expressing active AKT will be used to determine if rapamycin can inhibit or delay tumor formation. 4) Conduct a phase II trial of an mTOR inhibitor in the treatment of ovarian cancer. Overall, these studies will yield important insights regarding the value of AKT as a biomarker for predicting ovarian cancer development, progression and drug sensitivity and will ascertain whether AKT pathway inhibition can serve as an effective chemopreventive and/or chemotherapeutic strategy in ovarian cancer.
PROJECT 3 While advances have been made in understanding the biology and improving the treatment of ovarian cancer, improved diagnostic and therapeutic approaches are urgently needed. The ability of antibodies to target defined tumor-related structures can improve the selective identification and destruction of tumors. Recent advances in antibody engineering make it possible to structurally modify antibodies to optimize their tumor targeting and effector functions. For example, we have prepared and characterized a recombinant antibody-derived protein consisting primarily of antigen-combining sites (diabody) that targets HER2/neu. This diabody is a non-covalently joined single-chain Fv (scFv)-based dimmer that shows exceptional and unique promise the therapy of HER2/neu overexpressing malignancies. However, HER2/neu is infrequently overexpressed in ovarian cancer. Accordingly, it is necessary to consider different antigen targets to effectively exploit the potential of this novel antibody structural format. It is particularly desirable to target receptors in a manner that disrupts signaling for ovarian cancer growth and survival. The human Mullerian inhibiting substance (MIS) receptor is an attractive candidate target antigen in ovarian cancer. This receptor is expressed in a large percentage of ovarian cancer cell lines and cells isolated from ascites fluid collected from ovarian cancer patients. We have isolated the gene for the MIS type II receptor (MISIIR) extracellular domain (ECD) and have produced the ECD in mammalian cell lines. We hypothesize that high-affinity anti MISIIR diabodies will efficiently target ovarian cancer, and that these molecules will be effective delivery vehicles for therapeutic radionuclide applications. The aims of this proposal are: 1) To develop human diabody molecules specific for the extracellular domain of the MIS type II receptor; 2) To determine the in vivo targeting and anti-tumor properties of radiolabeled anti-MISIIR diabody molecules; 3) To perform pilot clinical trials that will determine the sensitivity and specificity of 124I-radiolabeled anti-MISIIR diabody-based targeting in ovarian cancer. By the end of the proposed funding period the data generated by these studies will support the initiation of a pilot, Phase I therapy trial employing a radiolabeled anti-MISIIR diabody for the treatment of ovarian cancer.
PROJECT 4 There is an immediate need for new, selective anticancer agents that differentiate between malignant and nonmalignant cells. The benefits of such agents would include a higher therapeutic index and lower toxicity than conventional therapies. Although expressed in nonmalignant cells, the epidermal growth factor receptor (EGFR) is highly expressed in a variety of tumors and its expression correlates with poor survival. Evidence for a role for the EGFR in the pathogenesis of various cancers, including ovarian cancer, has led to the rational design and development of agents that selectively target this receptor. Activation of the EGFR signaling pathway in cancer cells has been linked with increased cell proliferation, angiogenesis, and metastasis, and decreased apoptosis. Preclinical data show that anti-EGFR therapies can inhibit these effects in vitro and in vivo in several types of tumor cells. In addition, preclinical data confirm that many such agents have the potential to increase the effectiveness of current cytotoxic agents. Following accelerated drug development programs, phase II/III trials are now underway for a number of EGFR-targeted therapies, including monoclonal antibodies IMC-C225 (ErbituxTM, cetuximab), ABX-EGF, EMD-72000, and the EGFR-tyrosine kinase inhibitors gefitinib (ZD1839, IressaTM) and erlotinib (OSI-774, TarcevaTM OSI/Genentech). Thus, the rationale for EGFR-targeted approaches to cancer treatment is apparent and now well established, and there is increasing evidence that they may represent a significant contribution to cancer therapy. However, the efficacy of this targeted therapy has yet to be fully explored in ovarian cancer. Furthermore, it is not clear which components of this pathway are important in the therapeutic response and how maximum efficacy can be achieved. In the studies proposed, we will evaluate not only therapies directed against 170-kDa EGFR, but also an isoform of the receptor (EGFRvIII) that is expressed in a high percentage of solid tumors, including ovarian. An Inter-SPORE phase II clinical trail will evaluate the efficacy of cetuximab in patients with ovarian or primary peritoneal carcinoma who have persistent or recurrent disease following previous platinum/taxane-based chemotherapy. Cetuximab will be given at doses to achieve grade 1 rash, therapy assuring that a biologically active dose of cetuximab was administered. Integrated genomic and proteomic approaches will be used to identify components of this signaling pathway which are important in the therapeutic response. Finally, we will explore emerging therapies using a transgenic mouse model of ovarian cancer developed at our institute. We will evaluate the efficacy an EGFRvIII peptide vaccine, referred to as LEEK, in this in vivo model. The overall goal of this project is to evaluate current and developing molecular-targeted therapies, to improve the treatment of ovarian cancer, and ultimately to enhance frontline therapy increasing the possibility of cure.
PROJECT 5 The long-term survival for women with advanced ovarian cancer remains poor despite large improvements in the short-term survival. This proposal is poised to address the barriers to successful immunization of women with ovarian cancer, ideally, as they complete induction chemotherapy. Studies in our laboratory identified a T cell subset of CD4+25+ regulatory T cells (TREG) that exhibit potent immunosuppressive effects in the local tumor environment. These TREG represent a substantial proportion of tumor-infiltrating lymphocytes in some ovarian tumor specimens. We will also present evidence from our laboratories indicating that select women with advanced ovarian cancer have a potent form of tumor immunosurveillance at diagnosis that profoundly impact the natural course and clinical outcome of ovarian cancer, creating a strong rationale for implementing immunotherapy for this disease. Our findings indicate that in approximately half the patients with ovarian carcinoma, intra-tumoral T cells and/or tumor-reactive peripheral blood T cells mediate an anti-tumor immune response. Based on these findings, we hypothesize that tumor-infiltrating, tumor-associated or peripheral blood T cell can be used for the preparation of adoptive T cell therapy in select patients with ovarian carcinoma. Extensive preliminary data support the following three specific aims: In Aim 1, we will determine the therapeutic potential of tumor-associated T cells in ovarian cancer. Preliminary studies supporting this aim demonstrate that we have developed an innovative system of artificial antigen-presenting cells (aAPCs) allowing the exponential amplification of effector CTL that maintain antigen specificity, permitting the generation of clinically meaningful quantities of T cell for adoptive therapy. Work in Aim 1 is likely to generate novel approaches for adoptive immunotherapy with intra-tumoral T cells, and to characterize the effects of TREG on tumor infiltrating lymphocytes. In Aim 2, we will assess the effects of intra-tumoral dendritic cell injections on T cell effector functions in a syngeneic mouse model of ovarian carcinoma. In addition, experiments are proposed to dissect the effects of tumor-derived vascular endothelial growth factor (VEGF) on T cell mediated tumor immunity. In Aim 3, two clinical trials are proposed to validate experimental concepts derived from our laboratories. In the first trial, patients with ovarian cancer will be vaccinated with autologous mature dendritic cells loaded with peptides specific for human telomerase reverse transcriptase (hTERT) and her2/neu. In this trial we will determine if cyclophosphamide enhances the anti-tumor response, and if it depletes TREG cells. In the second trial, we will explore the potential of intra-tumoral DC injections to reverse local tumor-mediated immunosuppression. Thus, we are now poised to test our hypotheses that barriers erected by TREG and VEGF prevent patients from reaching their full response to therapeutic vaccination. This project will bring together a highly collaborative team of basic scientists and clinical investigators to enable novel trials with the goal of improving the long term outcome for ovarian carcinoma.
PROJECT 6 New approaches to early detection of ovarian cancer are urgently needed since existing surgical and management methods can consistently cure only early stage cancer. There is broad agreement that the genetic and epigenetic alterations which initiate and drive cancer can be potentially useful in the diagnosis and management of cancer. Silencing of tumor suppressor genes, such as p16INK4a, VHL and hMLH1, have established promoter hypermethylation as a common mechanism for tumor suppressor inactivation in human cancer cells. Hypermethylation has been shown to be a promising target for molecular detection of several types of cancer in bodily fluids, however ovarian cancer had not yet been tested. Using sensitive methylation specific PCR, we screened matched tumor, pre-operative serum or plasma and peritoneal fluid (washes or ascites) DNAs obtained from 50 patients with ovarian or primary peritoneal tumors for hypermethylation status of the normally unmethylated BRCA1 and RAS association domain family protein 1A (RASSF1A) tumor suppressor genes. Hypermethylation of one or both genes was found in 34 tumor DNAs (68%). Further examination of one or more of the adenomatous polyposis coli (APC), p14ARF, p16INK4a or death associated protein-kinase (DAP-Kinase) tumor suppressor genes revealed hypermethylation in each of the remaining 16 tumor DNAs which extended diagnostic coverage to 100%. Hypermethylation was observed in all histological cell types, grades and stages of ovarian tumor examined. An identical pattern of gene hypermethylation was found in the matched serum DNA from 41 of 50 patients (82% sensitivity) including 13 of 17 cases of stage I disease. Hypermethylation was detected in 28 of 30 peritoneal fluid DNAs from stage IC-IV patients, including 3 cases with negative or atypical cytology. In contrast, no hypermethylation was observed in non-neoplastic tissue, peritoneal fluid or serum from 40 control women (100% specificity). We have begun validation of this first study of methylation-based detection of ovarian cancer by examining futher ovarian cancer patients and controls and testing new quantitative real time MSP "Methyl-Light" technology. We have also performed a global epigenome drug-based reactivation screen of ovarian cancer cell lines with the aim that the genes identified will extend, and improve, our early detection panel to include epigenetic profiling, in regard to prognosis and behavior, of a particular tumor. We conclude that promoter hypermethylation is a common and relatively early event in ovarian tumorigenesis that can be detected in the serum DNA from patients with ovary-confined (stage IA or B) tumors and in cytologically negative peritoneal fluid. Analysis of tumor specific hypermethylation in serum DNA may enhance early detection of ovarian cancer.
CORE A The Administrative Core is responsible for providing scientific direction and leadership for the interdisciplinary Ovarian SPORE Proposal and for providing administrative support for all five Projects, five Core resources, the Developmental Research Program, and Career Development Program. The Administrative Core will ensure a continuing emphasis on translational research and will support the inter-SPORE collaborations with the other Ovarian SPORE institutions. This Core will be responsible for maintaining and expanding interactions between Fox Chase Cancer Center and the University of Pennsylvania. The Administrative Core will provide support for the functions of the Executive Committee, which will be responsible for continually evaluating the progress of the Projects, the status of the Pilot Projects, the efficient use of other shared resources (Cores), and monitoring the progress of Career Development awardees. The Administrative Core will assume overall fiscal and scientific responsibility, and specifically will focus on the use of SPORE flexibility in providing funding for programs and Pilot Projects based on the success or failure to meet translational research goals in ovarian cancer. The Ovarian SPORE Administrative Core will also coordinate and support the functions of the External Advisory Committee and the Internal Advisory Committee and coordinate all meetings, retreats, and external consultations. This Core is responsible for the bimonthly ovarian cancer seminar series. The Administrative Core will also maintain close consultation with NCI SPORE staff and will be responsible for all regulatory requirements and for preparation of non-competitive renewals.
CORE B This Core is intended to be a resource for the SPORE as a whole and a valuable source of biological material and genetic information for our continuing studies of ovarian cancer. The Biosample and Tissue Procurement Core (BTPC) will be responsible for collecting, banking, and distributing ovarian tumor specimens (fresh or paraffin embedded) accrued from patients treated surgically at FCCC. In addition, this Core will isolate nucleic acids and proteins from ovarian tumors and will construct, and update the tissue-microarray collection. Ovarian tumor specimens and prophylactic oophorectomy specimens will be banked and will be distributed according to specific requirements of SPORE projects. Furthermore, this Core will collect, process, and distribute blood-derived biosamples acquired at FCCC before and after surgical procedures, as well as biosamples of all available non-surgical patients, especially those undergoing prophylactic oophorectomy and individuals from families at high risk to contract ovarian cancer. Accompanying personal and family history information will also be obtained for each of these participants. The BTPC will also process blood collected through the "Ovarian Cancer Consortium for Research and Surveillance" Core. Biological samples collected at all the Ovarian Cancer Network sites will be forwarded to this Core for processing (e.g., extraction of genomic DNA) and banking (e.g., leukocytes, erythrocytes, plasma, and serum) under CLIA approved guidelines. Constitutive DNA isolated from a subset of these samples will be tested for germ-line mutations in BRCA1 and BRCA2 to help identify participants eligible for the proposed chemoprevention trial. The BRCA1 and BRCA2 mutation carrier status will ultimately be used to assess the effectiveness of bilateral prophylactic oophorectomy and/or chemoprevention agents at reducing the women's risk of developing ovarian cancer. The BPTC will also provide a comprehensive pathology and immunohistochemical support, both at the technical and interpretative levels for all of the SPORE projects and many of the pilot projects. The BTPC will work closely with Core E, "Biostatistics and Data Management Core" to insure that critical clinical information can be linked to each biosample. In addition to the guidelines of quality control (QC) established by the College of American Pathologist, the Core will follow strict specific rules of QC for each of its components. All operations, prioritization and distribution activities will be overseen by the SPORE Executive Committee. The BTPC will eliminate the redundant infrastructure and expense that would be required for each SPORE researcher to develop their own tissue bank or biosample repository allowing for more synergism and productivity.
CORE C The Ovarian Cancer Consortium for Research and Surveillance (OCCRS) Core has created a truly unique and dynamic resource of families who, in partnership with our scientific team, are committed to the prevention and control of ovarian cancer. The OCCRS has been a natural response to the rapidly growing body of genetic-based insights into the process of ovarian carcinogenesis, bringing basic scientists together with clinicians, public health advocates and lay consumers in a series of multidisciplinary research projects. At the core of this resource is the cohort of almost 800 participants from 400 families characterized by one or more cases of ovarian cancer, with extensive information on relevant medical, epidemiologic, and behavioral factors, detailed family histories, and the storage of blood and tissue specimens from members of these extended families. A significant proportion of the participants have undergone clinical genetic testing for BRCA1/2 mutation status. Critical to its success has been the development of a comprehensive information management system which links family data, exposure variables, clinical information, screening behaviors, biospecimen data and genetic information in a secure and efficient operating system. Education, counseling, and clinical management protocols have been developed in concert with all of the collaborating sites. An important outcome of the work to date has been the refinement of skills and expertise in recruitment, a major challenge particularly for cancer prevention and control research involving families with a genetic risk for cancer. As a result of this expertise, the OCCRS has facilitated the initiation of several research projects across several disciplines and has been successful in obtaining additional funding sources. The OCCRS will continue to recruit eligible families to the Core, with accompanying medical and risk factor data and blood and biospecimen samples. The Core will interact closely with the Biosample and Tissue Procurement Core and the Biostatistics and Data Management Cores. The OCCRS will make available expertise in recruitment strategies for all clinically-based projects, and will provide assistance with the ever growing regulatory responsibilities associated with human research, especially that of a genetic nature. This clinical resource will have an ever-expanding role in ongoing and new research projects, both at Fox Chase Cancer Center (FCCC) and within the inter-SPORE consortium, to contribute to the goals of the SPORE program to create and environment conducive to translational research in ovarian cancer. Dr. Mary Daly will continue in her leadership position and will work closely with other SPORE leaders to promote cancer prevention and control projects.
CORE D This Core will produce genetically engineered mice prone to develop ovarian cancer for all Fox Chase SPORE projects. It will also be available to investigators at other ovarian SPORE institutions. This resource makes available the expertise of the Mouse Models of Human Cancer Consortium of which the Core director is a member. This addresses the request of NCI director von Eschenbach to integrate the MMHCC components into SPORE programs. The mice produced will be used to study targeted therapy, detection, immunotherapy, drug resistance, and prevention of ovarian cancer. The mice will be created by implementing the following Specific Aims: 1) Provide the backbone reagents and expertise necessary to produce ovarian cancer prone genetically modified mice; 2) Express cDNA's, provided by SPORE investigators, in the ovarian surface epithelium of transgenic mice or transgenic ovarian tumors using promoters identified by the Core Director and his collaborators; 3) Determine if mice created in Specific Aims 1 and 2 have altered ovarian histology; 4) Use mice created in Specific Aims 1 and 2 to produce crosses prone to develop ovarian cancer or alter tumor resistance to therapy; 5) Evaluate the mice produced in Specific Aim 4 for pathological and immunohistochemical features characteristic of human ovarian cancer.
CORE E The goal of the Biostatistics and Data Management Core (BDMC) is to facilitate ovarian cancer research at Fox Chase Cancer Center (FCCC) by providing expert experimental design, data management, and data analysis support to SPORE scientists and clinicians. Members of the Core have substantial knowledge and experience in biostatistics and data management as applied to cancer research. This highly interactive group of biostatisticians and data management experts will serve all SPORE projects and cores and support future projects and pilot studies. The specific goals of the Biostatistics and Data Management Core are to: 1) coordinate and manage SPORE statistical activities to ensure that investigators have ready access to statistical support; 2) provide statistical design of experiments and studies, including research proposal development, sample size determination, randomization procedures, plans for interim reviews and final analysis; 3) assist with the writing of statistical components of manuscripts; 4) review, all studies involving human subjects,; 5) collaborate with the Protocol Office in the development of protocols and the monitoring and reporting of clinical data; 6) conduct biostatistical methodology research on practical problems arising in ovarian cancer research; 7) provide computer-based tools that facilitate the storage and retrieval of data generated in the proposed research, thereby creating and maintaining a centralized relational database that provides access to common resources and information; 8) ensure the accuracy of the data maintained in the database by software-based data consistency and quality control systems; 9) organize and maintain the database to maximize accuracy and accessibility; while maintaining strict confidentiality; 10) provide detailed descriptions of available populations and resources for current and future SPORE investigators; and 11) provide data entry services. |
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