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Fred Hutchinson Cancer Research Center/ University of Washington
Overall Abstract The Pacific Northwest Prostate Cancer SPORE is a coordinated effort of four institutions with strong- programs in prostate cancer research and career development: 1) the Fred Hutchinson Cancer Research Center (FHCRC); 2) the University of Washington (UW) and its affiliated institutions; 3) the Institute for Systems Biology (ISB); and 4) the University of British Columbia and the Prostate Center & Institute of Vancouver General Hospital. These three Seattle-based and the British Columbia (BC)-based institutions have a large number of investigators and laboratories dedicated to prostate cancer (CaP) research. Within this milieu, there already exists substantial technical infrastructure ( e.g., genomics ), strong multidisciplinary expertise ( e.g., molecular biology, biochemistry, epidemiology, genetics, medical & radiation oncology, urology), and extensive resources (e.g., serum, DNA & tissue banks, CaP animal model facilities, CaP genomic arrays, and a CaP clinical trials organization). All four institutions have, and are in the process of generating, increasing resources for programs in translational CaP research and are committed to contributing significant resources toward the goals of this SPORE. The purpose of the SPORE is not only to perform the research projects proposed, but in a larger sense to form the "central supporting piece" to a large developing "mosaic" of coordinated translational CaP research in the Pacific Northwest. We believe the projects to be innovative and translational. Project 1 is a population-based study evaluating specific genetic polymoIphisms in relation to CaP progression/mortality .Project 2 aims to characterize molecular alterations (karyotype, transcript) of disseminated CaP cells in the context of influencing the clinical management of patients as diagnostic and prognostic indicators. Project 3 will identify critical determinants of the transition from androgen-dependent to androgen-independent CaP, and will validate these findings in well-characterized pre-clinical models and clinical trials. Project 4 seeks to expand our already extensive work on the genomics of CaP and will determine if tumor gene expression profiles can predict the course of disease and response to cytotoxic chemotherapy. We have proposed five Cores in support of these projects (Administration, Specimen/Tissue, Biostatistics, Informatics & Gene Expression, and Clinical Research). The Career Development and Pilot Project Programs we propose will significantly embellish and strengthen the translational orientation of our prostate cancer research and expand opportunities for new investigators. Project 1: Prostate cancer (CaP) is the most frequently diagnosed solid tumor and the second leading cause of cancer deaths among U.S. men. Despite the substantial morbidity and mortality associated with CaP, limited attention has been focused on elucidation of factors that predict clinical disease progression or mortality. Environmental and genetic factors may play a role in the development of aggressive, life-threatening CaP. We hypothesize that variant alleles in genes involved in the androgen/growth factor/antioxidant pathways confer a higher risk for disease progression and mortality. Specifically, we propose to genotype a population-based cohort of CaP patients to address the following hypotheses:
The study will take advantage of extensive interview and clinical data previously collected on 753 CaP patients diagnosed between 1993-1996 and followed for 10-13 years (through 2006). Stored genomic DNA (n=625) will be used for genetic analysis of these patients. Follow-up patient surveys, physician surveys, medical record reviews, and linkage with the Seattle SEER cancer registry will be used to identify patients with disease progression or death (all cause, prostate cancer-specific). Cox proportional hazards models will be used to assess genotype-CaP outcomes. As secondary aims, we will: 1) assess each measure of clinical progression (symptomatic, radiologic, biochemical) by genotype; 2) assess whether clinical or pathologic tumor characteristics at diagnosis vary by genotype; and, 3) utilize the host/lifestyle/environmental data to examine whether CaP outcomes vary by factors such as family history of prostate cancer, body mass index, or dietary intake. This study may identify genetic variants that are useful for the stratification of CaP patients into high-risk progression/mortality subgroups who may thus benefit from aggressive therapy and clinical trials of new adjuvant therapies. Results may also increase our understanding of the underlying molecular biology and genetic epidemiology of this common, life-threatening disease.
Project 2: From 20-40% of men who undergo a radical prostatectomy for localized disease later relapse with progressive disease that invariably results in bone metastases. Despite enormous efforts in the analysis of the primary tumor, few prognostic markers have been revealed. We have focused attention on the detection and isolation of disseminated CaP cells in the blood and bone marrow (BM). Our belief is that these disseminated cells, particularly those in the BM, may provide critical insight to the processes of progression. It would appear logical that the CaP cells in the BM that remain following a radical prostatectomy may be more informative than those of the primary tumor which is removed at surgery. Yet, the detection of these cells and especially their recovery for study has been heretofore most challenging. Our group has devoted a considerable effort to both of these tasks and this proposal is the result of successes in these endeavors. Our data reveal that approximately 65% of patients who undergo a radical prostatectomy have disseminated PSA+ cells in their BM prior to surgery. In this proposal we will expand our studies from the simple detection of disseminated cells to their characterization. In addition to standard immunohistochemistry, the analyses will utilize state-of-art techniques in gene expression micro-arrays and array Comparative Genomic Hybridization (array-CGH). We will use these approaches to test three major hypotheses: (1) Disseminated CaP cells isolated from the BM at the time of radical prostatectomy will reveal biological features useful in assessing the probability of relapse. These features will become evident by a comprehensive comparison of the results of whole-genome scans for karyotypic alterations and changes in gene expression levels in pools of disseminated cells isolated from patients at different stages of progression. (2) Residual disseminated CaP cells in the BM will provide more insight into recurrences than the primary tumor that is removed at surgery. Comparisons of karyotypic and gene expression profiles of bone metastases within and among patients will reveal common alterations in molecular pathways relevant to progression and growth in bone. (3) The characteristics of disseminated cells, particularly those related to gene expression levels, will modulate as a result of treatment. These studies will provide the first multiparametric analyses of disseminated cells that takes advantage of advances in (a) the isolation and recovery of disseminated CaP cells from bone marrow, (b) cutting edge technologies in expression micro-arrays and array-CGH, and (c) an extensive and comprehensive SPORE infrastructure that will provide the critical statistical and informatics support.
Project 3: Androgen ablation is the only form of systemic therapy demonstrated to prolong life in men with advanced prostate cancer (CaP), precipitating apoptosis in subpopulations of cells. Despite high initial response rates, remissions are temporary because surviving CaP cells usually recur with an androgen independent (AI) phenotype. Thus, one of the main obstacles to a cure of advanced CaP by androgen ablation is AI progression, a complex process involving variable combinations of clonal selection, adaptive upregulation of cell survival genes, and ligand-independent androgen receptor or alternative growth factor activation. We hypothesize that changes in apoptosis-associated gene expression play critical roles in tumor progression and drug resistance and that adaptive changes in gene expression after androgen withdrawal increase CaP cell survival, accelerate AI progression, and render cells chemoresistant. Identification and functional characterization of molecular events that allow cancer cells to survive treatments that normally induces apoptosis will improve our understanding of progression and identify new therapeutic targets. Application of microarray gene expression analysis to complimentary Shionogi and LNCaP model systems after apoptotic triggers like castration or chemotherapy will serve as a primary screen to identify apoptosis-associated gene clusters. Advantages of determining gene expression profiles in these well-defined xenograft models, compared to direct hybridization of human CaP tissues, include their predictability and reproducibility, circumventing inherent problems of small quantity of cancer typically present in regressed CaP specimens after androgen withdrawal and the heterogeneity of specimens from individual patients. Apoptosis-associated genes will undergo secondary screening in human CaP tissue arrays (comprised of cores from hormone naïve; 1, 2, 3, and 8 months after neoadjuvant hormone therapy (NHT); androgen independent; and post NHT + Taxotere) to verify relevance to the human disease. Candidate genes with putative pro-or anti-apoptotic functions will then be subjected to tertiary analysis for functional characterization in xenograft models (ie overexpression vs antisense-inhibition) or transgenic knockouts to clarify role in apoptosis and hormone resistance. As examples, in this proposal we plan to extend on our previous experience with Bcl-2, clusterin, IGFBP-2 and IGFBP-5 genes and characterize functional roles of 2 apoptosis-associated genes that increase after castration in Shionogi tumors - Hsp27 and p35 (regulator of cdk5). Finally, as an illustrative example of quaternary analysis, we will initiate a clinical trial using ASO's to target clusterin, an anti-apoptosis gene that we have recently shown in primary, secondary, and tertiary screens to increase after cell death triggers and enhance cell survival. We will evaluate the safety and activity of a 2nd generation antisense oligonucleotide targeting clusterin in a Phase I/II presurgical pharmacokinetic and pharmacodynamic study in men with localized prostate cancer. Collectively, these objectives will improve our understanding of apoptosis, adaptive cell survival mechanisms, drug resistance, and identify new therapeutic targets for clinical development.
Project 4: Prostate cancer is a complex disease that presumably stratifies into different types of cancers with distinct progression rates, metastatic potentials and prognostic outcomes. We hypothesize that histologically similar primary prostate carcinomas can be stratified into distinct categories of clinical behavior based upon profiles of their expressed genes. We will determine the gene expression profiles of prostate carcinoma that correlate with clinical phenotype of progression or non-progression as determined by PSA-free survival at 5 years post-therapy. The expression profiles from the normal and neoplastic tissues of 100 individuals that have undergone radical prostatectomy will be determined. Outcomes will be measured by following serial PSA measurements for recurrent disease. Correlations of individual and cohorts of genes with outcome will be determined using statistical analyses. Independent predictors of recurrence such as PSA and Gleason Grade will be evaluated as co-variables. We will also determine the molecular indicators of response to chemotherapeutic intervention for high-risk prostate cancers. The gene expression profiles of prostate tissue obtained pre- and post-neoadjuvant chemotherapy will be determined. Correlations between individual genes as well as multiple genes will be examined for association with a) histological measures of response; b) PSA response immediately post-therapy; and c) time to recurrence as determined by PSA-free survival at 5 years post-therapy. The advancement in high throughput technology such as cDNA microarrays and MS/MS technology allows one to use a systems approach to study human diseases rather than analyzing one gene or one protein at a time; that is to completely define the transcriptome (identify all mRNAs) and define much of the proteome (identify all proteins) of prostate cancer cells. We will apply high throughput technology and discovery-driven approaches to define the differences in transcriptomes and proteomes of the androgen-dependent LNCaP cell and its androgen-independent variant CL1 by 1) expression profiling using cDNA microarray containing 46k cDNAs, 2) MPSST (Massively Parallel Signature Sequencing) technology to determine about 1 million sequences from each cell line, and 3) proteomics ICAT labeling coupled with MS/MS. We will then evaluate the findings from cell line models with 40 tumor biopsies each from androgen-dependent prostate carcinoma and hormone-refractory using real-time quantitative RT-PCR, quantitative Western blot analysis and immunohistochemical analysis with tissue microarrays.
Core A: The Administrative Core will consist of a variety of interacting committees working closely with the PI (Paul Lange, M.D.) and Co-PI (Janet Stanford, Ph.D.) and the administrative staff consisting of trained personnel in finance, grants management, clinical research support, and educational/editorial services (see diagram following). The committees will involve:
Core B: Principal Investigator(s): Robert L. Vessella, Ph.D. The Specimen Core provides part of the infrastructure support for Projects 1 - 4 as well as for future pilot and developmental projects. It has been designed to meet the needs of these projects plus serve as a stand-alone resource for collaborative efforts with other SPOREs. This Core will provide a well-organized and standardized system of specimen collection, storage, distribution and related clinical/research information dissemination that is based on over two decades of experience. There will be consistency and quality assurance in the pathological analysis of tissue specimens. Furthermore, the centralized management of the specimens will facilitate their distribution based upon the priorities defined by a panel of investigators familiar with all of our SPORE research endeavors. There are five central components to this Core:
An administrative program that includes a proactive program of obtaining specimens from minorities, a specimen utilization plan, a quality control program and a program for interactions between SPOREs.
Core C:
Biostatistics The Biostatistics Core will play a pivotal role in the Northwest Prostate Cancer SPORE, interacting with virtually every project and Core. The defining goal of the Biostatistics Core will be to assist investigators in formulating studies that can feasibly address the questions of scientific interest, are amenable to statistical analysis, and will ultimately yield statistically valid and interpretable results. This Core will link study design, data collection, measurement, and analysis to the critical hypotheses and questions of the Northwest Prostate Cancer SPORE through the following Specific Aims:
To implement analyses of genomic expression data, the Biostatistics Core will utilize a software system for analysis of genomic expression data, currently under development at the Fred Hutchinson Cancer Research Center. This system will provide researchers on the SPORE with a user-friendly interface to a variety of standard as well as novel computational algorithms for the analysis of expression data. The Biostatistics Core will work closely with the Informatics and Gene Expression Core to define the microarray database for the array expression data generated by the proposed SPORE, and to select procedures for data normalization and quality control. The Biostatistics Core will also work with the Clinical Research Core to define the structure of a new clinical database, to design clinical trials of new treatment strategies and to design clinical validation studies for confirming the importance of genes discovered through SPORE projects.
Core D: The Informatics and Gene Expression Core (IGEC) serves to provide resources and perform services for SPORE Projects I-IV as well as for future developmental and pilot projects. The IGEC will facilitate the accomplishment and integration of SPORE research objectives involving studies of large-scale gene expression in a timely and cost efficient manner, and will also serve as a resource for collaborative studies and technology evaluation and dissemination with other SPOREs. A major activity of the IGEC will be the construction and analysis of specialized cDNA arrays for large-scale transcript expression studies. We are well positioned to provide this resource by virtue of our efforts to develop a prostate-centric gene expression orientation with the following resources:
The IGEC will provide:
Database archiving of IGEC data for secure storage with rapid Website access/dissemination to Projects and collaborators. (Conjunction with Cores B and C).
Core E: Principal Investigator(s): Celestia S. Higano, M.D. Successful translational research depends of the flow of information not only from the laboratory to the clinic but also from the clinic to the laboratory and back. The objectives of the Clinical Core are:
To support a translational research infrastructure. Members of Core E will play an active role in the Translational Working Teams (TWT) which will meet every other month. The ultimate goal of these teams is to bring promising research findings into the clinic as quickly as possible. Core E will design and conduct clinical trials which grow out of TWT meetings. The multi-disciplinary, translational approach will also be used as a model to train fellows in clinical research, specifically in prostate cancer.
Developmental Research Program The purpose of the Developmental Research Program (DRP) of this Pacific Northwest Prostate Cancer (CaP) SPORE is to support innovative translational CaP research projects aimed at reducing CaP morbidity and mortality and improving survival of CaP patients. This program will provide a mechanism to quickly respond to new translational research opportunities that may develop within the SPORE environments and its four participating institutions; that is, in BC, Canada, the Vancouver General Hospital (VGH), Prostate Centre; and in Seattle, the University of Washington (UW), Fred Hutchinson Cancer Research Center (FHCRC), and the Institute for Systems Biology (ISB). Such support will allow innovative pilot projects to mature sufficiently so that they can successfully compete for additional funding from sources either within or outside the SPORE. This program will also recruit different scientific expertise and new investigators in translational CaP research. The specific aims are: 1) To provide pilot funds for 1-2 years for innovative investigator-initiated ideas in all areas and 2) To allow the SPORE leadership to target funds to specific areas that are especially likely to advance the translational research goals of the SPORE. An aggressive solicitation of proposals will be made inside our SPORE affiliated institutions one year before funding starts. The Project Leader of the proposed project must possess either an MD, MD/PhD or a PhD equivalent in a related area of clinical, population, or laboratory research. They must be a member of one of the SPORE institutions. They must agree to participate in all the SPORE-related educational and communication activities and have the potential and inclination to continue in translational CaP research after the project ends. A 10 member committee will select 3-6 proposals per year for funding using a rigorous system of evaluation. Funding will usually be for one year. The Project Leader of the funded project will be fully integrated into the administrative and education activities of the SPORE. Significant institutional support has been directed to this program. We have already solicited proposals, received 11, and present 6 herein as examples of the quality, diversity, and potential of such a program in our environment.
Career Development Program A Career Development program will be a major and essential part of this proposed SPORE. Its goal will be to develop physicians and basic scientists for lifelong productive careers in translational prostate cancer research. The program will strive to make these individuals scientifically productive, academically successful and influential nationally, and good role models for recruitment of individuals to similar careers. The program will involve all of the institutions within the SPORE in Seattle (FHCRC, UW, Institute for Systems Biology) and BC (UBC and Prostate Center at VGH). The coordination between the two areas with be primarily with regard to recruitment, educational opportunities, and the interchange of mentors and research opportunities. However, proposed SPORE funding for this program will concentrate primarily in Seattle. The specific goals of this program are to:
Recruitment of fellows and staff will occur through the planned educational program and by systematic recruitment activities. These individuals will be monitored by a specific Career Development committee, members of the SPORE, and by over 33 non-SPORE senior investigators whose research interest and accomplishments reflect prostate cancer research concerns. In addition to SPORE grant support, this program will be supported by extensive institutional resources. |
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