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2005 Scientific Report

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Laboratory of Cancer

Laboratory of Cancer Immunodiagnostics Brian B. Haab, Ph.D. Dr. Haab obtained his Ph.D. in chemistry from the University of California at Berkeley in 1998. He then served as a postdoctoral fellow in the laboratory of Patrick Brown in the Department of Biochemistry at Stanford University. Dr. Haab joined VARI as a Special Program Investigator in May 2000. Staff Songming Chen, Ph.D. Michael Shafer, Ph.D. Sara Forrester, B.S. Darren Hamelinck, B.S. Randall Orchekowski, B.S. Laboratory Members Students Thomas LaRoche Richard Schildhouse Research Interests Many cancers are difficult to detect at early stages and are often diagnosed too late to allow curative treatment. Earlier and more accurate detection of cancer could lead to better outcomes for many patients. A greater knowledge of the molecular changes associated with the development of cancer could lead to a better understanding of disease mechanisms and improved diagnostic tests. The Haab laboratory is developing novel experimental approaches to gather such molecular information and to use it for the diagnosis of cancer. Our goal is that these studies will produce measurable benefits to cancer patients. We are taking a variety of approaches to identify changes in blood protein composition that define cancer and that could be diagnostically useful. Microarray methods have features that are particularly useful for this research. We have been developing several related antibody and protein microarray methods—such as two-color competition assays, sandwich assays, glycan detection, and antigen detection of antibodies—to analyze serum samples from cancer patients and controls. With these methods (Fig. 1), we can efficiently probe the binding to many different antibodies and proteins and explore the use of multiple measurements for classifying samples. Previous technological developments that are now in routine use include a high-sensitivity detection method (two-color rolling-circle amplification) and a new method for isolating multiple microarrays on a single slide, allowing highthroughput sample processing. Our research also makes use of gel and chromatographic separations and mass spectrometry to provide complementary experimental information. An ongoing study of protein profiles from the sera of pancreatic cancer patients and controls (in collaboration with Randall Brand and George Vande Woude) shows the value of antibody microarrays for diagnostics research. Protein profiles from antibody microarrays targeting a wide variety of proteins—such as those previously associated with cancer, involved in biological systems altered by cancer, or having elevated levels in the tumor environment—are revealing many proteins at either higher or lower abundances in the cancer patients. Some of the proteins were not known to be associated with pancreatic cancer and may contribute to improved early detection of the disease. The serum samples can be classified as from cancer or control patients using the antibody measurements. The accuracy of the classifications was greatly improved with multiple measurements in combination (relative to using individual measurements); we need to further develop this approach for cancer diagnostics. A recent modification to this technology measures alterations in the glycosylation state of the proteins. Glycosylation—the attachment of specific carbohydrate structures to proteins— plays a major role in determining protein function, and glycosylation alterations have been associated with the development and progression of cancer. The ability to conveniently measure changes in specific carbohydrates on different proteins could be valuable in identifying the changes most 28

associated with cancer and thus of use for diagnostics. We have methods for detecting changes in glycosylation on proteins captured by antibody microarrays (see Fig. 1C) and are using those methods to profile the glycosylation alterations on serum proteins from cancer patients and control subjects. We use protein microarrays (see Fig. 1D) to gather additional information about changes occurring in the blood of cancer patients. Some tumor proteins elicit the production of antibodies targeting those proteins. The identification and measurement of tumor-recognizing antibodies could provide information about molecular alterations in the tumor and be valuable in cancer detection. The protein microarray is an ideal screening tool for that purpose. In collaboration with Gilbert Omenn and Samir Hanash, microarrays of tumor-derived proteins from cancer cell lines are probed with sera from cancer patients to identify proteins recognized by the patients’ antibodies. An ongoing study of serum from prostate cancer patients (in collaboration with Alan Partin) has identified several proteins that could be involved in an immune response. We are characterizing the nature of the responses and the proteins involved. The above methods may be applied in a novel way to further their effectiveness. We have begun to develop and improve existing diagnostic markers through the use of longitudinal measurements (serial measurements over time). In a collaboration with William Catalona, Robert Vessella, and Ziding Feng, we are looking at changes over time in the concentrations of several serum proteins leading up to disease recurrence in prostate cancer patients. We hypothesize that the use of individualized thresholds defining abnormal protein levels, defined by each person’s history of measurements, will yield improved diagnostic accuracy over the use of single, population-wide thresholds. Our hypothesis has been supported in some individual demonstrations, and we now have an experimental system for systematically exploring it for a large number of proteins and many patients. Figure 1. Antibody and protein microarray formats. A) Two-color competition assay. Two pools of proteins, respectively labeled with biotin and digoxigenin tags, are mixed and co-incubated on antibody microarrays. The relative amount of binding to each antibody from the two pools is determined through detection of the biotin and digoxigenin tags. B) Sandwich assay. After incubation of a protein sample on an antibody microarray, the amount of protein binding to each antibody is measured using a second antibody that targets the captured proteins. C) Glycan detection. A pool of digoxigenin-labeled proteins is incubated on antibody microarrays, and the level of protein binding at each antibody is determined by detection of the digoxigenin tag. The amount of a particular glycan on the captured proteins is detected using a biotin-labeled protein that specifically binds to that glycan. D) Protein array detection of antibodies. Serum samples are incubated on arrays containing a variety of tumor-derived proteins. Antibodies in the serum samples that recognize and bind to the proteins are detected using a secondary antibody that binds to human antibodies. We are seeking to apply the methods described above in ways that will have a positive result in terms of cancer care. The incorporation of mass spectrometry methods, through collaborations with Greg Cavey at VARI and members of the Michigan Proteome Consortium, will provide more opportunities for discovery. The further testing of the value of these tools is being pursued through local clinical oncology programs. The access to clinical practice is especially valuable for translating the development and discovery in our laboratory into benefits for cancer patients. External Collaborators Phil Andrews, Gilbert Omenn, and Diane Simeone, University of Michigan, Ann Arbor Randall Brand, Evanston Northwestern Healthcare, Illinois E. Brian Butler and Bin S. Teh, Baylor College of Medicine, Houston, Texas William Catalona, John Grayhack, and Anthony Schaeffer, Northwestern University, Evanston, Illinois 29

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