8:30-8:35 Chairperson’s Opening Remarks

8:35-9:00 Chips to Kits: Innovation from Biomarker Discovery to Commercial Diagnostics
Brian T. Edmonds, Ph.D., Principal Investigator, Manager New Technologies, Integrative Biology & Global Discovery Research, Eli Lilly & Co. 
The advent of the era of personalized medicine increases the need to push protein biomarkers from preclinical research into clinical validation and finally to commercialization. With complex diseases, biomarkers increasingly will be discovered and assayed using novel technologies. Yet, even with validation of clinical utility, the logistic challenge remains of obtaining buy-in on this innovation from the entire stakeholder value chain. If the purposes of pharma, payers, patients, regulatory, and diagnostics are misaligned, the true medical benefit of personalized medicine will never be realized. The presentation will highlight case studies of our experience navigating protein biomarkers through the process of discovery to FDA-approved test.

9:00-9:25 iMASS² – A Novel Peptide Chip Technology for Clinical Diagnostics
Christoph H. Borchers, Ph.D., Department of Biochemistry and Microbiology, Director, Proteomics Centre, University of Victoria
Mass spectrometry (MS)-based protein chip technologies are useful tools for high-throughput determination of protein expression levels and protein modification status from complex mixtures. Here we discuss some applications of iMASS2, our novel MS-peptide chip technology based upon immunoaffinity capture of proteolytic peptides from proteins of interest, to provide highly-sensitive, specific, and quantitative determination of protein expression levels and phosphorylation status in clinical samples. We have used the iMASS² approach to determine bacterial proteins such as p23 from Francisella tularensis, and to determine EGFR from cancer cells (including biopsy samples). To achieve the highest possible sensitivity in these assays, we used customized antibodies that had been raised against p23 and EGFR peptides, which had been selected because of their high sensitivity in MALDI-MS. Under these optimal conditions we could detect these peptides at attomole levels, and demonstrated a dynamic range for absolute quantitation of greater than two orders of magnitude. This is equivalent to a protein concentration of femtograms/mL and a detection limit of EGFR in a few tens to one hundred cells.

9:25-9:50 Diagnostic Applications for Protein Biomarkers: The Application of Single Parameter, Multi-Parameter, and Multiplexed Biomarker Assays in Diagnostics and Targeted Therapy
Thomas M. Grogan, M.D., Professor of Pathology, University of Arizona and Chief Scientific Officer, Ventana Medical Systems, Inc.
Through a combination of single-analyte protein and multi-parameter gene and protein biomarkers, as well as through the use of multiplexed assays, we are defining a toolkit that allows for more accurate diagnosis and treatment of cancer. A case in point for the single analyte biomarker is the rabbit monoclonal antibody detection of cKIT in gastrointestinal stromal tumors (GIST). In this case, single analyte specificity can be effective to the 97 percentile, as confirmed by mutational analysis. In another circumstance, e.g. Herceptin to treat Her2-positive breast cancer, it may be necessary to use a combined gene and protein multiparameter assay. In this instance, a combination of in-situ hybridization and immunohistochemistry fully articulates the relationship between gene and protein expression and may be pivotal in explaining responders to therapy. Finally, the prediction of patient response to targeted therapy may require a multi-analyte analysis. In particular, response to therapy for the Her2 gene family may necessitate investigating a matrix of the downstream signaling molecules. In the case of multiple analytes being analyzed simultaneously, the next-generation of fluorochromes, specifically conjugated Quantum Dots, is required along with image analysis. This combination of multiplex QDot and image analysis may be said to comprise flow cytometry on a glass slide. In these examples, protein biomarkers are a critical part of the diagnostic tool kit and empower decisions leading to targeted therapy.

9:50-10:15 Immunological Assay Platform Based on Spinning Disc Interferometry
Fred Regnier, Ph.D., J.H. Law Distinguished Professor, Department of Chemistry, Purdue University
Disease associated protein biomarkers are being discovered at an ever increasing rate. But before they can be used clinically it is necessary to establish their diagnostic utility. Unfortunately, throughput and quantification with current mass spectrometry based biomarker discovery methods will not accommodate the requisite 103 – 104 patient samples used in validation trials. This presentation will discuss a new protein array system that uses spinning disc interferometry (SDI) to evaluate protein binding at 26,000 or more immobilized protein array elements in a few min without the need for secondary reagents. The immobilized proteins are generally antibodies, but can be any type of binding protein. When used to conduct immunological assays the current generation of discs have up to 260 sample “wells” with 100 array elements per well. Protein array elements are 100 um in diameter, but can be smaller when there is need to add still more array elements per well. After robotic addition of 15 uL samples to wells on the disc, samples are incubated for sufficient time to allow complex formation. The disc is then spun rapidly and washed to remove unbound proteins. Dried discs are then transferred to the SDI reader where a disc is spun at 6,000 rpm while array elements are being read multiple times. Current sensitivity is 50 pg of protein antigen per mL of sample but detection limits of 1 pg/mL have been achieved.

10:15-11:00 Coffee Break with Exhibit and Poster Viewing

Biomarkers to Predict Response to Therapy

11:00-11:25 Identification of Phosphoprotein Theranostic Markers for Patient Tailored Medicine and Carrier Protein Bound Peptidome Markers for Disease Detection
Emanuel F. Petricoin, Ph.D., Co-Director, Center for Applied Proteomics and Molecular Medicine, Chair, Department of Molecular and Microbiology, George Mason University
Our laboratory has developed a series of enabling proteomic technologies and methods for tissue-based theranostic markers and blood borne markers for early stage disease detection. The reverse phase protein microarray, a new type of proteomic technology, provides a highly multiplexed profiling of kinase-driven cellular signaling networks from human biopsy specimens. Case studies that illustrate the utility of this technology for clinical applications will be presented. Carrier protein bound biomarkers, arising from a complex information archive comprised by the low molecular weight peptidome, are currently being discovered. We describe this novel finding along with the development of an immuno-mass spectrometry platform that can readily distinguish both the size and identity of the candidates.

11:25-11:50 Circulating Oncoproteins as Cancer Biomarkers
Walter Carney, Ph.D., President, Oncogene Science, Biomarker Development, Bayer Healthcare
Oncogene Science, which is a center for Oncology expertise at Bayer Healthcare, is developing Biomarkers to go with targeted therapies. We have developed a panel of 4 oncoproteins that are circulating in patients with cancer and have clinical value in conjunction with targeted therapies directed to the oncoproteins HER-2, EGFR, MN and ras. Case studies will be presented for the serum HER-2/neu test in HER-2 positive breast cancers and how levels of this oncoprotein correlate with clinical outcomes to various therapies. Biomarker data will also be presented for EGFR, ras and MN, all circulating Oncoprotiens. Biomarker data will be shown correlating circulating VEGF-165 levels with clinical outcomes in renal cell carcinoma patients treated with the Multi-kinase inhibitor, Nexavar.

11:50-12:15 Mass Spectrometry Identification of Pathways and Biomarkers Predictive of Treatment Outcome
John Haley, Ph.D., Senior Fellow, Translational Research, OSI Pharmaceuticals Inc.
The molecular determinants which confer cell sensitivity to molecularly targeted agents in tumors have been unclear. Quantitative measurement of epitheial-mesenchymal transition markers in human tumor specimens will be correlated with treatment outcome in patients receiving EGFR and IGF1R tyrosine kinase inhibitors. The identification of treatment biomarkers was readily achieved by a rapid and quantitative stable isotope peptide labeling approach coupled to LC-MS/MS.

12:15-1:45 Lunch on Your Own

Proteomic Data Analysis for Biomarker Identification

1:45-2:10 A Comparison of Proteomic Biomarkers and a Proteomic Fingerprint
Brian T. Luke, Ph.D., Senior Scientist, Advanced Biomedical Computing Center, SAIC-Frederick/NCI Frederick
While there is a great deal of discussion about proteomic biomarkers, most analyses of MS, LC/MS, 2D-NMR, and other spectra construct classifiers based on proteomic fingerprints. Biomarkers and fingerprints are extremely different, mainly in the assumption of the number of specific states (think genotypes) associated with each category (phenotype). Identifying the underlying protein product is important with the biomarker method, while the fingerprint method has to be concerned with complete coverage of all possible states and the uniqueness/validity of the proposed classifier. While it is possible to consider the entire fingerprint as a single biomarker, researchers who construct classifiers based on fingerprints incorrectly state that the peaks included in the fingerprint represent a group of biomarkers (or “panel of markers”). There needs to be a fundamental understanding of the difference between a biomarker and a fingerprint, and this talk concentrates on this comparison.

2:10-2:35 Interpretation of Quantitative Shotgun Proteomic Data
Alexey I. Nesvizhskii, Ph.D., Assistant Professor, Department of Pathology, University of Michigan
The shotgun proteomic strategy is being increasingly applied to the expression profiling of human samples. However, the peptide-centric nature of shotgun proteomics complicates the analysis and biological interpretation of the data. The same peptide sequence can be present in multiple different proteins or protein isoforms, which leads to ambiguities in determining the identities of sample proteins. In this talk we will discuss the difficulties of interpreting shotgun data and describe transparent informatics approaches that address them. We also discuss related issues such as the current state of protein sequence databases and their role in shotgun proteomic analysis, interpretation of relative peptide quantification data in the presence of multiple protein isoforms, the integration of proteomic and transcriptional data, and the development of a computational infrastructure for the integration and data mining of multiple diverse datasets.

2:35-3:05 Toward the Targeted Detection and Quantitation of the Interferon Alphas in Serum by Mass Spectrometry
Dr. David Arnott, Protein Chemistry, Genentech Inc.

3:05-3:45 Refreshment Break with Exhibit and Poster Viewing

Post-Translational Modifications as Biomarkers