Wednesday, April 11
Proteomic Sample Preparation

7:30 am Registration and Morning Coffee

8:30 Opening Introductions

10:00 Coffee Break

10:35 Emergent Technology for Proteomic Biomarker Discovery from Formalin-Fixed Paraffin-Embedded Tissue
Brian L. Hood, Ph.D., Research Scientist, Clinical Proteomics Facility, University of Pittsburgh Cancer Institute
Formalin-fixed paraffin-embedded (FFPE) tissues are often overlooked for mass spectrometry-based tissue biomarker discovery investigations as they are considered intractable to standard proteomic methods of analysis. We have recently developed a simple methodology for efficient extraction of unmodified peptides from FFPE tissue and incorporated this novel technology in a comprehensive tissue biomarker discovery workflow. This emergent tissue disease biomarker discovery workflow will be discussed in the context of its application to cancers of the prostate, head and neck, and breast.

11:05 The Use of Fresh Human Tissue in Drug Discovery Research
James Eliason, CSO, Research and Development, Asterand, Inc.; Associate Professor, Barbara Ann Karmanos Cancer Institute, Wayne State University
Genomic and proteomic techniques have increased the importance of human tissue in drug discovery research. Much focus has been made on use of snap frozen tissues, but fresh tissues play an important role as well. Fresh viable tissues not only can be used for proteomic and genomic studies but provide the basis for functional studies that test the efficacy and toxicity of lead compounds.

11:35 Emerging Platforms (Sponsorship Available)

12:05 Lunch Workshop or Lunch on your own (Sponsorship for Technology Presentation Available)

1:25 Chairperson’s Remarks

1:30 Solutions to Insoluble Problems: Exploring the Synergy of Hydrostatic Pressure and Chemistry for Biological Sample Preparation
Alexander V. Lazarev, Ph.D., Director, Research & Development, Pressure BioSciences, Inc.
Tremendous progress in analytical instrumentation development for the past several decades has brought powerful tools like mass spectrometry, quantitative PCR, and automated sequencing into most laboratories. To the contrary, many popular sample preparation approaches present considerable limitations, potentially leading to the lack or reproducibility or losses of important analytes. This presentation will focus on applications of alternating hydrostatic pressure as a novel sample preparation technique orthogonal to chemical or enzymatic lysis or mechanical homogenization. Pressure Cycling Technology (PCT) exhibits synergy with temperature and many chemical reagents. Optimized combination of these approaches allows nearly complete dissolution of relevant biological material from a variety of historically problematic samples. Examples of PCT applications for genomics, proteomics, drug metabolism and protein purification will be presented. 

2:00 Towards Comprehensive Proteomic Mapping of Blood Vessels through Prefractionation and Multi-Modal MS Analysis 
Jan Schnitzer, M.D., Scientific Director, Department of Cell Biology, Sidney Kimmel Cancer Center
Tissues are very complex with extensive molecular diversity over a wide concentration range that creates challenges for proteomic analysis. To reduce data complexity and dynamic range limitations, we subfractionate human and rodent tissue samples to isolate specific endothelial plasma membranes of interest using specialized colloidal nanoparticle coating technique. The proteins are analyzed either directly by digestion, HPLC of peptides and tandem mass spectrometry or after protein separation by gel electrophoresis. This multi-modal approach along with multiple measurements per sample expands protein coverage and detection several fold. Examples of newly identified tissue-specific blood vessel targets will be presented along with validation studies using anitbodies and molecular imaging to demonstrate specific target expression as well as specific organ and solid tumor immunotargeting in vivo. This strategy illustrates the discovery power of combining proteomics & subfractionation to unmask novel targets impossible to detect by current standard techniques.

3:00 Meet-the-Experts  Brainstorming discussion groups moderated by experts in the area. Attendees are invited to choose the table according to their main interest; however, they may switch between roundtables. We emphasize that this roundtable discussion is for an interactive exchange among scientists and is not meant to be, in any way, a corporate or product discussion. Topics moderated: Table 1: The Importance of Protein Partitioning and Fractionation: Enrichment before Downstream Analyses Host: TBA To partition or not to partition? That is the question.”  Specificity & reproducibility? Methods for high capacity proteome fractionation Digging into the proteome using these tools. How deep can you go? Table 2: Gel Based Proteomic versus Peptide Labeling Proteomic Host: Sunny Tam, Ph.D., Director of Proteomic Fractionation Group, Research Associate Professor, UMMS Proteomic Consortium, University of Massachusetts Medical School Pros and cons of 2D gel versus iTRAQ  What are the expected outcomes of the two approaches?  What are the future improvements for the two approaches? Table 3: Biorepositories/HIPAA/IRB Host: James L. Wittliff, Ph.D., M.D. hc, Professor of Biochemistry & Molecular Biology, James Graham Brown Cancer Center, University of Louisville Table 4: Influence of Sample Collection Parameters on Functional Assays Host: Scott D. Patterson, Ph.D., Senior Director, Medical Sciences, Amgen Inc. Which analytes change rapidly following collection?  What realistic controls can be put in place?  Validity of ex vivo stimulation as oppose  Table 5: RNA Amplification and Labeling for Array Profiling  Host: Christoph Adams, Ph.D., Research Area Manager/Epigenetics, Life Sciences Division, Invitrogen Corporation Normalizing RNA sample input  What constitute proper internal controls for array normalization? When is RNA amplification a good idea?  How many microRNAs are there in humans?  Table 6: Fresh versus Frozen Tissue in Drug Discovery Research Host: James Eliason, CSO, Research and Development, Asterand, Inc.; Associate Professor, Barbara Ann Karmanos Cancer Institute, Wayne State University Table 7: Sample Collection and Processing, a Pivotal Stage of Transcript Profiling Host: Eric R. Fedyk, Ph.D., Senior Scientist II, Drug Safety Evaluation, Millennium Pharmaceuticals, Inc.  When to collect and store samples frozen versus formalin-fixed and paraffin-embedded?  Isolate subpopulations of leukocytes or store whole blood?  Optimal method of RNA isolation for specific sample types, tissues, throughput (capacity), etc.?  What are the most predictive quality control metrics for mRNA, miRNAs, rRNAs, tRNAs, etc.?  What are the most predictive quality control metrics for cDNA, cRNA and/or qPCR reactions

4:30 Discovery of Antecedent Biomarkers for Alzheimer’s Disease
Douglas A. Hinerfeld, Ph.D., Research Assistant Professor, Proteomic Fractionation Group, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School
A number of laboratories have examined the cerebral spinal fluid (CSF) from patients with either minimal cognitive impairment (MCI) or Alzheimer’s Disease (AD) in the attempt to identify protein biomarkers that are indicative of disease. While meeting with much success, these studies have been limited by the fact that the disease process has already progressed to the point of producing symptoms, which may be too late to begin effective treatments as the neuronal loss is already significant. In our laboratory, we have access to CSF from subjects that are presymptomatic for AD but which, due to a mutation in the presinilin 1 (PS-1) gene, are likely to incur symptoms within the next decade. We have exploited state-of-the-art proteomic technologies, including pre-fractionation, differential 2D gel, and mass spectrometry-based approaches to identify proteins that are modified in the CSF of the presymptomatic PS-1 subjects that would be indicative of the earliest stages of the disease. Using these global proteomic techniques, combined with targeted analysis of CSF, we have identified a number of proteins whose concentrations are altered in the CSF of the presymptomatic PS-1 subjects. 

5:00 Selection of Highly Informative Subproteomes for Biomarker Discovery
David M. Smalley, Ph.D., Assistant Professor, Mellon Medical Biomarker Discovery Laboratory, University of Virginia
Most current biomarker discovery efforts focus on the ability to fractionate complex protein mixtures using analytical techniques and to analyze everything. In contrast, our research has examined subproteomes of urine and plasma that have a high likelihood of containing biomarkers. Such proteomes include microparticles from plasma, exosomes from urine, and the phosphoproteome from bladder cancer cell lines.

5:30 End of Day One

Thursday, April 12
Proteomic and Genomic Sample Preparation

8:30 Chairperson’s Opening Remarks

10:00 Grand Opening Refreshment Break in Exhibit Hall

11:00 Chairperson’s Remarks

11:05 Gene Expression Profiling from Difficult Samples such as FFPE
Gianfranco de Feo, Ph.D., Senior Director, Customer Solutions, NuGEN Technologies
Access to biologically and clinically relevant samples for the discovery, refinement, and validation of gene expression signatures reflecting clinical phenotypes has been hindered by the inability to generate high quality gene expression results from the most readily available tissue sources, formalin fixed, paraffin embedded tissues. NuGEN technologies has recently developed a linear, isothermal, and robust amplification approach to perform whole transcript amplification from very small amounts of total RNA (below 5ng input). 
This technology will not only allow researchers to perform splice variant analysis, but will also allow for the use of degraded RNA samples, such as RNA isolated from FFPE sources, in expression experiments. Data demonstrating the performance of the technology, including sensitivity, linearity, dynamic range, and differential expression accuracy will be shown. In addition, data demonstrating the performance of the approach to clinically relevant samples such as RNA isolated from FFPE tissue sources will also be shown.

11:35 Measurement of Gene Expression from Fixed Tissue: qNPA Validation of Biomarkers for Diffuse Large-B-Cell Lymphoma
Bruce Seligmann, Ph.D., Chairman & CSO, HTG, Inc.
Measurement of gene expression from fixed tissue using the lysis only qNPA multiplexed ArrayPlate assay measures the total RNA in the tissue including the “in situ” cross-linked mRNA. It is sensitive (using only 1/4th of a tissue slice), gives equivalent quantitative results as measurement from fresh or frozen tissue, and gene expression levels correlate with their protein product biomarkers measured in situ by immunohistochemistry. Three independent studies using snap frozen tissue had identified non-overlapping sets of biomarker genes, for a total of 36 putative biomarkers. All 36 were measured plus cell lineage genes. The validation of a biomarker set and correlation to therapeutic response and survival outcome will be reported.
These results demonstrate that archives of fixed tissue can now be easily mined for biomarker and target validation. Safety studies, clinical development, and diagnostic assays can be performed using fixed tissue without changing current tissue storage practice, to get gene expression results that correlate to protein biomarkers without any pre-qNPA sample prep.

12:45 Lunch on your own (Luncheon Seminar Sponsorship Available) 

 (Sponsorship Available)
Selected leaders and technology providers in the field will explain their technology and equipment in detail and the attendees will have the opportunity for a “close-up-and-personal” hands on experience. Plenty of time for individual questions will be given.

3:30 Refreshment Break in Exhibit Hall

4:15 The Investigation and Use of Microheterogeneity in Human Plasma Proteins
Randall W. Nelson, Ph.D., The Biodesign Institute, Arizona State University
This presentation will focus on the investigation of microheterogeneity -- e.g., point mutations, slice variants or post-translational modifications -- occurring in human plasma proteins. Using mass spectrometric immunoassay, microheterogeneity is observed, characterized and cataloged in moderate-sized cohorts (100’s - to - 1,000’s) of healthy individuals. Microheterogeneity in diseased cohorts is characterized in the same manner, and molecular variants considered indicative of disease are subsequently used in the systematic development of multiplexed mass spectrometric immunoassays able to detect disease with high accuracy. Examples will be given for the investigation of cardiovascular disease.

4:45 Optimizing the Detection of DNA Based Biomarkers in Blood Plasma
Theo deVos, Ph.D., Vice President, Diagnostic Development, Senior Scientist, Epigenomics Inc.
Measurement of genetic and/or epigenetic markers in DNA from plasma or serum has great potential for the development of screening tests in oncology. To realize this potential, sample collection and processing must be optimized to measure low levels of a target biomarker in a background of normal DNA. We present our efforts to optimize collection and storage of plasma samples, as well as our workflow for sample processing and analysis of DNA based biomarkers. As a case study, we describe our experience validating Septin 9 DNA methylation as a plasma biomarker for colorectal cancer.

5:15 Molecular Profiling of Circulating Tumor Cells – RNA Preparation, Microarray Protocols and Fish Analysis
Mark Connelly, Ph.D., Vice President, Department of Reagent Development, Immunicon
Circulating tumor cells (CTCs) can be detected in blood from patients with metastatic and primary carcinomas. Over the past several years, the development of CellTracks® technology based on immunomagnetic capture and fluorescent characterization of CTCs has enabled accurate enumeration of CTCs at extremely low frequencies, on the order of 1 CTC per 7.5 ml of whole blood. Pivotal clinical trials have shown that the number of CTCs before treatment is an independent predictor of progression-free and overall survival in patients with metastatic breast cancer. Interim analyses of data from clinical trials in prostate and colorectal cancer appear to mirror data from the breast cancer trial. The talk will focus on antisense RNA library construction from CTCs obtained from hormone-refractory prostate cancer patients, as well as global gene expression profiling of CTCs and Circulating Endothelial Cells. FISH analysis and protocols will be also discussed.

5:45 Networking Reception in Exhibit Hall

7:00 End of Day Two