Ovary – Fred Hutchinson Cancer Research Center (Coordinating Center)
| Project Title: | Pacific Ovarian Cancer Research Consortium | |
| Cancer Site: | Ovary | |
| Principal Investigator(s): | Nicole URBAN, Sc.D. | |
| Participating Institutions: |
Fred Hutchinson Cancer Research Center (FHCRC – Coordinating Center)
University of Washington Swedish Medical Center Marsha Rivkin Center for Ovarian Cancer Research Benaroya Research Institute at Virginia Mason Institute for Systems Biology Cedars Sinai Medical Center BC Cancer Agency |
|
| Clinical Samples: | Human tissues, sera, plasma, and urine | |
| Technical Approaches: |
Transcript and Proteomic Technologies
ELISA Assay ScFv Yeast Display Library Bead-based Multiplexing Assay System Protein/peptide quantitation using visible isotope-coded affinity tags (VICAT) and 16/18O-labeling |
Brief Description of Project:
The Pacific Ovarian Cancer Research Consortium (POCRC) SPORE in ovarian cancer brings together a group of dedicated and enthusiastic scientists from 8 institutions, each one contributing substantially to the intellectual activity of the SPORE. Institutions in the POCRC include the University of Washington (UW), Swedish Medical Center (SMC), Cedars-Sinai Medical Center (Cedars), Institute for Systems Biology, (ISB), Marsha Rivkin at Virginia Mason as well as the FHCRC and the Vancouver Island Cancer Center (VICC) in British Columbia. The Center for Ovarian Cancer Research (MRC), and Benaroya Research Institute POCRC has identified four areas where opportunities exist for us to make a difference, including the identification of tumor markers and the subsequent development of blood tests for risk assessment and early detection of ovarian cancer.
Early detection, a key to improved outcomes for women with ovarian cancer, is a major focus of our research program. Our general strategy in meeting translational research goals includes 1) exploitation of emerging molecular technologies to identify biologically relevant genes, proteins and antigens as candidate markers and targets for translation, 2) a systematic approach to prioritizing markers and targets for evaluation, 3) a collaborative approach to evaluating candidate markers and targets that includes evaluation of candidates identified by colleagues at other institutions, and 4) use of novel statistical methods to use markers to predict biologic phenotype.
Our primary objective is to recommend a set of markers and an algorithm that can be used in a clinical trial for ovarian cancer screening to prevent ovarian cancer from escaping early detection. Achieving this goal requires the development of a novel statistical model to identify promising markers, as well as the evaluation of several currently existing and novel markers using serum specimens from two currently funded studies for ovarian cancer screening.
We have developed statistical methods for combining markers in a panel [1], and for using biomarkers longitudinally to improve their performance [2]. These methods are useful for evaluation of the markers for inclusion in the panel, as well as for using the panel clinically. We are evaluating the protein products of several genes that we found earlier to be over-expressed in malignancy [3], for their presence in serum and for their ability to identify women with ovarian cancer. Selection of the genes for study was based on statistical analysis of their joint contribution to a panel able to discriminate between malignant and normal tissue. Top genes include HE4, Mesothelin, SPINT2, SLPI, CD24, IFI27, MUC1, Keratin 8, FOLR and GPR39. Using mouse monoclonal antibodies, sandwich ELISA's were developed for HE4 and Mesothelin. To validate their performance singly and in a panel, proteins were measured by ELISA in an independent set of blinded samples. HE4 was shown to be more specific than CA125 in discriminating women with malignant tumors from those with benign tumors [4]. Mesothelin in combination with CA125 was shown to perform better than either used alone in discriminating women with malignant tumors from healthy women [5].
To complete the panel, we are using novel affinity reagents. Antibodies are the classical reagents used to detect disease markers in a large variety of diagnostic tests, from IHC to ELISA tests. Single chain Fragment variable (scFv) antibodies contain only the heavy and light chain variable sequences of the antibody recognition sequences. To isolate such antibodies, we are using the yeast display scFv library developed by Feldhaus and colleagues [6], which contains 2x109 scFv derived from a naïve human B cell population. The yeast display scFv are tagged with HA and c-myc epitopes, and recognize proteins without regard to their immunogenicity. We use the library both to identify scFv against known proteins such as SPINT2 and SLPI, and for discovery of novel proteins. For the latter application, we proceed by several rounds of subtraction as for cDNA subtractive libraries. The yeast display scFv library has several advantages over other available sources of antibodies such as the phage display libraries. First, it features the absence of any growth-related bias, due to a galactose promoter that prevents scFv expression during library amplifications. In addition, it provides the ability to sort the specific antigen-binding scFv by flow cytometry, and the efficient production by the yeasts of the scFv for future use. We have isolated one scFv candidate that binds preferentially to a pool of ovarian carcinoma sera versus a pool of control sera, and have identified the corresponding protein by immunoprecipitation and mass spectrometry. As in any proteomics approach, the source of patient material is critical, as it determines the discriminatory power of the antigen(s) identified.
- McIntosh MW and Pepe M, Combining Several Screening Tests: Optimality of the Risk Score. Biometrics, 2002. 58: p. 657—664.
- McIntosh MW and Urban N, A Parametric Empirical Bayes Method for Screening using Longitudinal Observations of a Biomarker. Biostatistics, 2003. 4(1): p. 27—40.
- Schummer M, Ng WV, Bumgarner RE, Nelson PS, Schummer B, Bednarski DW, Hassell L, Baldwin RL, Karlan BY, and Hood L, Comparative hybridization of an array of 21,500 ovarian cDNAs for the discovery of genes overexpressed in ovarian carcinomas. Gene, 1999. 238(2): p. 375-85.
- Hellstrom I, Raycraft J, Hayden-Ledbetter M, Ledbetter JA, Schummer M, McIntosh M, Drescher C, Urban N, and Hellstrom KE, The HE4 (WFDC2) protein is a biomarker for ovarian carcinoma. Cancer Research, 2003. 63(13): p. 3695-700.
- McIntosh M, Drescher C, Karlan B, Scholler N, Hellstrom K, and Hellstrom I, Combining CA 125 and SMR serum markers for diagnosis and early detection of ovarian carcinoma. Gynecologic Oncology, 2004. 95(1): p. 9-15.
- Feldhaus MJ, Siegel RW, Opresko LK, Coleman JR, Feldhaus JM, Yeung YA, Cochran JR, Heinzelman P, Colby D, Swers J, Graff C, Wiley HS, and Wittrup KD, Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nat Biotechnol, 2003. 21(2): p. 163-70.
Team Members and Expertise:
Martin MCINTOSH, Ph.D. (FHCRC – Statistician and Project Principal Investigator) [mmcintos@fhcrc.org]
Dr. McIntosh is an associate member at Fred Hutchinson Cancer Research Center (FHCRC), where in addition to his research activities he is the director of the FHCRC comparative proteomics group. He is also an associate professor at the University of Washington in the Department of Biostatistics. He has served as an external advisor for early detection programs at the CDC, the Early Detection Research Network (EDRN), the National Cancer Institute, and the ovarian cancer program at the M.D. Anderson Cancer Research Center. He is also a founding member of the U.S. HUPO Council. Dr. McIntosh is the Principal Investigator of the POCRC's early detection project and is also a co-investigator on a department of defense center of excellence (COE) grant (Dr. Nicole Urban PI) for evaluating biomarkers for breast cancer, and is co-principle investigator of a NCI funded R01 for identifying molecular profiles that can detect cervical cancer and predict cervical cancer progression.
Dr. McIntosh has researched early detection of cancer, particularly using biomarkers, for over five years, and has authored or co-authored several publications on screening trials evaluation, biomarkers, algorithms for cancer screening, cost effectiveness, and algorithms for constructing biomarker. His technical area of expertise is in evaluating the effectiveness of population based screening programs; methods to combine markers identified using high throughput methodologies, and using novel (longitudinal) screening algorithms in experimental screening studies. Dr. McIntosh leads the technical aspects of fitting screening algorithms and heads the committees that monitor the performance of the markers in the POCRC early detection work.
Nicole URBAN, ScD. (FHCRC – POCRC Principal Investigator and Early Detection Project Co-Investigator) [nurban@fhcrc.org]
Dr. Urban is a Member at Fred Hutchinson Cancer Research Center, and an expert in health services, especially in the area of screening for cancer. She is PI on several research grants dealing with ovarian cancer on topics that include behavioral/psychosocial interventions, micro simulation modeling, and molecular biology. Dr. Urban has expertise in quantitative research and substantial knowledge about cancer screening policy and biology.
Garnet ANDERSON, Ph.D. (FHCRC – Project Co-Investigator) [garnet@whi.org]
Dr. Anderson is a biostatistician and current chair of the National Ovarian Inter SPORE Informatics Committees. She is also Co-PI for the Clinical Coordinating Center of the Women's Health Initiative (WHI) and is responsible for data operations, data management, computing and statistics for this study. She is a former chair of the WHI Steering Committee. Dr. Anderson currently chairs the WHI Design and Analysis Committee, which is responsible for the review of all ancillary studies to WHI and for determining investigator access to the WHI specimen repository. Dr. Anderson is the Principal Investigator of the ovarian cancer risk project. Dr. Anderson serves as the lead for all analyses of the WHI data.
Natalie SCHOLLER, M.D., Ph.D. (FHCRC – Co-Investigator) [nscholle@fhcrc.org]
Dr. Scholler is an immunologist who is presently a senior staff scientist at the FHCRC, where she is developing assays for novel early detection genes. She has extensive experience from Seattle Genetics and Pacific Northwest Research Institute (PNRI). Dr. Scholler is the lead author of the manuscript developing the mesothelin/MPF marker. She develops novel assays for this project from proteins identified from the developmental research projects.
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