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Overview Pre-Conference EventsRegister PDF DownloadConference-at-a GlanceDistinguished FacultyPosters Hotel & Travel Sponsor / ExhibitPress Pass Request Brochure
Lead Sponsoring Publications
7:00 -12:00 Registration for Pre-Conference Events
8:00-11:00 Pre-Conference Tutorial* (*Separate Registration Required)
Instructors: Jean Lee, Ph.D., Scientific Director, PKDM, Amgen Inc.; and Viswanath Devanarayan, Ph.D., Director, Statistics, Biomarker Research, Abbott Laboratories
This tutorial will provide recommendations on the “fit-for-purpose” best practices in the development and validation of biomarker assays and sample analysis. Special emphasis will be on assays where a reference standard material for the biomarker analyte is available. We will provide an overview of the key elements in the broad roadmap to method development and validation for the intended exploratory or advanced biomarker applications. The special challenges in protein biomarker assays will be discussed, including sample stability and collection integrity, validation and QC samples, validity of reference standards, calibration curve fitting methods, method optimization and method feasibility studies. Strategies for moving from biomarker panels in the exploratory phase to the markers chosen to support clinical trials will then be discussed from the analytical perspective. Finally, the recommendations for pre-study and in-study validation will be provided with brief illustrations.
8:00-11:00 Pre-Conference Workshop* (*Separate Registration Required)
8:00-8:30 Non-Coding RNAs as Cancer BiomarkersGeorge A. Calin, M.D., Ph.D., Associate Professor, Experimental Therapeutics & Cancer Genetics, The University of Texas, M. D. Anderson Cancer Center
MicroRNAs were linked to the progression of all types of human tumors that were investigated to date. The main molecular alterations are represented by variations in gene expression, usually mild and with consequences for a vast number of target protein coding genes. Recent studies proved that miRNAs are main candidates for the elusive class of cancer predisposing genes and that other types of non-coding RNAs participate in the genetic puzzle giving rise to the malignant phenotype. These discoveries could be exploited for the development of useful markers for diagnosis and prognosis, as well as for the development of new RNA-based cancer therapies.
8:30-9:00 miRNAs as Novel Biomarkers for Detecting Cervical Cancer Lymph Node MetastasisSylvie Beaudenon, Ph.D., Senior Scientist, Asuragen, Inc.
miRNA expression profiles can distinguish between lymph nodes, normal cervix and cervical squamous cell carcinomas. miRNAs are differentially expressed between cancer-positive and cancer-negative inguinal lymph nodes from cervical cancer patients. We have identified a subset of seven miRNAs that can distinguish positive from negative lymph nodes from cervical cancer patients. These miRNAs may represent new diagnostic markers for the detection of lymph node metastases in cervical cancer patients.
9:00-9:30 Development of miRNA-Based Biomarkers for Cancer Treatment SelectionAdam Baker, Ph.D., Director, Diagnostic Product Development, Exiqon A/S
Abnormal expression of microRNAs (miRNAs) in cancer implies that these small ~22-nucleotide molecules play a role in oncogenesis. Therefore miRNAs may comprise a novel class of diagnostic and prognostic signatures. This talk will focus on examples of using microRNA for cancer classification, prognosis and treatment selection.
9:30-10:00 Coffee Break
10:00-10:30 Her2/neu, microRNAs and HerceptinMichael N. Liebman, Ph.D., Senior Institute Fellow, Windber Research Institute; Managing Director, Strategic Medicine, Inc.
Herceptin treatment in breast cancer requires the observance of overexpression of Her2/neu in the patient, as measured by FISH and/or IHC. Only 25% of all patients overexpress Her2/neu, and only 40% of these patients respond to Herceptin. In collaboration with BIOBASE, we have pursued upstream analysis of the observed gene expression differences in patients where FISH and IHC present different results and have determined that microRNA appears to function as a switch in determining the differential response. This has been analyzed in terms of its potential use as a diagnostic and/or therapeutic target to improve decision-making for treatment in breast cancer patients.
10:30-11:00 Evaluation of microRNAs for Diagnosis and Prognosis of Patients with Solid TumorsMitch Raponi, Ph.D., Principal Research Scientist, Biomarkers, Centocor Research & Development
MicroRNAs (miRNA) are short non-coding RNAs that control the expression of multiple proteins through various mechanisms. It has been shown that miRNAs are differentially expressed in different cancers and limited functional studies have implicated specific miRNAs as either oncogenes or tumor suppressors. Using various expression profiling technologies we have identified and evaluated miRNAs that are potential diagnostic and prognostic biomarkers for colorectal cancer (CRC) and squamous cell lung cancer, respectively. In addition, in vitro functional studies of selected miRNAs have provided insight into their biochemical role in CRC. The utility of these analytes compared to previously defined mRNA classifiers will be discussed.
8:00-3:00 Pre-Conference Workshop* (*Separate Registration Required)
8:00-8:30 A Novel Label-Free Differential Analysis Work-Flow for Protein Biomarker Discovery from Tumor Tissue Interstitial Fluid –
Speeding Translation of Disease Marker Identification to AssayThomas P. Conrads, Ph.D., Associate Professor, Department of Pharmacology & Chemical Biology; Co-Director, Clinical Proteomics Facility, University of Pittsburgh Cancer Institute, Magee-Women’s Research Institute
Conventional protein biomarker discovery investigations are predominantly performed with samples such as serum or plasma. While serum or plasma is more desirable from a clinical standpoint, tissue likely possesses a greater abundance of readily identifiable proteins directly reflective of disease. Although many disease relevant proteins may be identifiable from tissue, most of these proteins are unlikely to be released from the tissue into the circulatory system, thereby limiting their clinical utility. We propose that tissue interstitial fluid (TIF), a proximal fluid that bathes cells, may provide a novel connection between tissue and serum to permit the identification of proteins that posses a high likelihood of being directly related to the disease process and that are readily assayable from serum. We have developed a detailed workflow that employs Fourier transform (FT) mass spectrometry (MS) and label-free quantification for biomarker investigations from TIF harvested from the renal cell carcinoma (RCC) tumor microenvironment. This workflow involves dissection of tumor and adjacent normal kidney (ANK) tissue from radical nephrectomies less than 10 minutes after surgical resection. Thereafter, these tissues are diced into 1 mm3 sections, placed in PBS and allowed to incubate for 1 hr at 37 ˚C in a 5% CO2 atmosphere. The TIF supernatants are collected and an equivalent amount of protein is digested and analyzed by liquid chromatography coupled online with an Orbitrap MS. Robust differential peptide abundance analysis is performed using a statistics-based differential expression algorithm (SIEVE), followed by directed feature (frame) identification using SEQUEST. Our results demonstrate the ability to identify and quantify proteins involved in cell-cell and integrin signaling, along with many cancer-related proteins, directly from the tumor microenvironment. This workflow enables direct identification and quantification of shed and secreted proteins from the tissue microenvironment and will speed translation to blood-based biomarker assays.
8:30-9:00 Data-Independent LC-MS/MS Profiling of Unfractionated SerumDave Goodlett, Ph.D., Professor, Medicinal Chemistry, University of Washington
We specialize in shotgun proteomic characterization of unfractionated cell lysates via gas-phase fractionation and label-free quantification. To understand i) what is detectable in a complex proteomic mixture with and without solution-based fractionation and ii) when/if to pre-fractionate prior to LC-MS/MS, we carried out exhaustive data-independent (DI) analysis of a well characterized bacterial lysate and human serum immuno-depleted of the top seven most abundant proteins. Collision induced dissociation (CID) of unique 1.5 m/z ranges was carried out in DI mode covering 400-1400 u. For 67 triplicate (automated) LC-MS/MS analyses 15 m/z were examined per injection without precursor ion selection similar to “shotgun” CID, except that an LTQOT was used. DI analysis of P. aeruginosa lysate identified more than 1650 unique proteins with an average protein sequence coverage of 33% without accounting for accidental CID and results from serum analysis show more than 300 proteins identified, casting doubt on the value of pre-fractionation.
9:00-9:30 New Paradigm in Proteomics Biomarker Discovery and EvaluationBruno Domon, Ph.D., Group Leader, Institute of Molecular Systems Biology, Swiss Federal Institute of Technology, Zurich
Proteomics undergoes a paradigm shift towards targeted strategies to overcome some of the limitations of conventional approaches. Hypothesis-driven, SRM based analysis of proteotypic peptides (surrogate for the targeted proteins) enables measurements over a wide dynamic range in complex samples. The SRM technique with two levels of mass selection provides high structural selectivity and unmatched sensitivity.
9:30-10:00 Current Status of Protein Biomarkers: What We Have Learned and What Lies AheadSamir Hanash, Ph.D., Program Head, Molecular Diagnostics, Public Health Sciences, Fred Hutchinson Cancer Research CenterAlthough our understanding of the molecular pathogenesis of common types of cancers has improved considerably, the development of effective strategies for cancer diagnosis and treatment have lagged behind. The vast dynamic range of protein abundance in plasma and the likely occurrence of tumor derived proteins in the lower range of protein abundance represent a major challenge in applying a proteomic based strategy for their identification. A combination of innovative strategies promises to overcome these challenges. Recent experience in comprehensive profiling of plasma proteins indicates that low abundance proteins may be identified and quantified with high confidence following extensive plasma fractionation and with the use of protein tagging procedures and high-resoltuion mass spectrometry. From an experimental design point of view, most cancer biomarker studies, including those aimed at identifying markers for early detection, are initiated with analysis of specimens from newly diagnosed subjects. The preferred approach for discovery of such markers is to utilize plasma obtained at a pre-clinical stage, prior to the diagnosis of cancer. Another implementation of this approach is through the use of mouse models of cancer that potentially represent an efficient means for uncovering diagnostic markers because of the ability to engineer mice that harbor genetic alterations known to be associated with tumors in humans, and because of the limited heterogeneity among mice bred under uniform conditions and the ability to sample blood in a standardized manner, at defined stages of tumor development. The current status of these approaches will be presented.
10:00-10:30 Coffee Break
10:30-11:00 MS-Based Quantitative Proteomics for Clinical DiagnosisChristoph Borchers, Ph.D., Associate Professor, Biochemistry & Microbiology, University of Victoria; Director, UVic–Genome BC Proteomics Centre
A particular focus of our group is the development of MS based techniques such as Multiple Reaction Monitoring (MRM) and immuno-MALDI towards their use in clinical proteomics for diagnostics. Both techniques are enabling us to provide absolute quantitation (concentration vs. up/down-regulation) through the use of isotopically-labeled peptides spiked in as internal standards. While MRM assays are capable for quantifying high to moderate abundant proteins, iMALDI is particularly useful for low abundant proteins. We have developed these assays for simultaneous quantitation of the 45 most abundant proteins in blood and cancer-related proteins like EGFR in clinical specimen including biopsy samples.\
11:00-11:30 High-Throughput Cell-Based Studies and Protein Microarrays for Biomarker and Target DiscoveryJoshua LaBaer, M.D., Ph.D., Director, Institute of Proteomics, Harvard Medical School
We developed a novel form of protein microarray, called nucleic acid programmable protein array (NAPPA), which can be used to study protein-protein interactions, protein-drug interactions, search for enzyme substrates, and as tools to search for disease biomarkers. In particular, recent experiments have focused on using these protein microarrays to search for autoantibody responses in cancer patients. Several bona fide autoantibody responses, such as responses to p53, have been detected, and a pilot study of responses to 7500 full length human proteins in 50 breast cancer patients and 50 controls has identified over 700 candidate proteins with more frequent responses in patients. These experiments show promise in finding antibody responses that appear in only cancer patients.
11:30-12:00 Brain Damage Markers: From Discovery in the CSF to Validation in the PlasmaJean-Charles Sanchez, Ph.D., Head, Biomedical Proteomics Group, Faculty of Medicine, Geneva University
Following any form of brain insult, proteins are released from damaged tissues into the cerebrospinal fluid (CSF). This body fluid is therefore an ideal sample to use in the search for biomarkers of neurodegenerative disorders and brain damage. We used human post-mortem CSF as a model of massive brain injury and cell death for the identification of such protein markers. Pooled post-mortem CSF samples were analyzed using a protocol that combined immunoaffinity depletion of abundant CSF proteins, isobaric mass tagging by the Tandem Mass Tags® (TMT®) technology, 2DLC and protein identification and quantitation by LC-MS/MS. A total of more than 400 proteins were identified, of which 250 proteins were not previously described to be present in CSF. Of these 250 proteins, more than 75% have been described as intracellular proteins suggesting that they were released from damaged cells. From these proteins, five have been further validated as biomarkers in age/sex matched European and American cohorts for the early diagnosis of stroke (UFD1, NDKA and DJ1), the thrombolytic treatment follow-up of ischemic stroke (GSTP) and the prediction of poor outcome after Subarachnoid Aneurysmal Hemorrhage (a panel of markers). Taken together, the validation in plasma samples of proteins found differentially expressed by TMT® in post-mortem CSF samples by proteomics approaches demonstrated the value of this concept as a first step toward the discovery of blood markers of brain injury.
12:00-12:45 Lunch on your own
12:45-1:15 Differential Protein Expression Analysis Using Spectral Count Data in Label-Free Shotgun ProteomicsAlexey I. Nesvizhskii, Ph.D., Assistant Professor, Department of Pathology, University of Michigan
Spectral counting has become a popular approach for measuring protein abundance in label-free shotgun proteomics. At the same time, the development of data analysis methods has lagged behind, and previously established methods for gene expression microarray experiments cannot be effectively applied. We present a set of computational tools for statistically robust analysis of spectral count data. We demonstrated that spectrum counting can be used as a quick and effective approach for uncovering up-regulated biological functions and pathways in both cell line and patient tissue profiling studies.
1:15-1:45 Mass Informatics of Mass Spectrometry-Based Protein Biomarker DiscoveryXiang Zhang, Ph.D., Associate Professor, Analytical Chemistry, University of Louisville
We have developed the Proteome Discovery Pipeline (PDP), a stand-alone bioinformatics platform used for LC/MS data analysis and biomarker discovery. Data is processed in a series of self-contained analytical steps using modules that are controlled by a graphical user interface. Modules included are spectrum deconvolution, alignment, normalization, significance tests, pattern recognition and molecular correlation networks. Modules consist of applications developed in C++ and the MATLAB which are called as external processes from the GUI using inputted parameters. Molecular correlation analysis is viewed using an interactive visual data mining approach.
1:45-2:00 Refreshment Break
2:00-2:30 Proteomic Mapping of Vascular Biomarkers to Image Antibody Penetration into Single Organs and Solid TumorsJan E. Schnitzer, M.D., Scientific Director, Professor of Cellular & Molecular Biology, Director of Vascular Biology & Angiogenesis Program, Sidney Kimmel Cancer Center
Enhancing noninvasive imaging and pharmacodelivery by targeting disease biomarkers is challenged by in vivo barriers limiting their access. Blood vessel endothelium prevents tissue penetration of many imaging agents, drugs, nanoparticles and gene vectors. Our discovery and validation strategies integrate tissue subfractionation, subtractive proteomics, bioinformatic interrogation, antibody generation, expression profiling, and various imaging modalities to quickly identify the in vivo targetable subset of biomarkers. Mapping proteins at endothelial cell surfaces in tissue yield novel vascular biomarkers enabling tissue- and disease-specific immunotargeting in vivo. This “organellar proteomic imaging of organ and disease biomarker space” creates opportunities for many diseases.
2:30-3:00 The Application of MALDI Mass Spectrometry Imaging (MALDI-MSI) to the Clinical Management of Prostate CancerO. John Semmes, Ph.D., Anthem Professor for Cancer Research; Director, Center for Biomedical Proteomics, Eastern Virginia Medical School
Diagnosis of prostate cancer is based on pathologic morphologic evaluation. The ability to localize disease-specific molecular changes in tissue would help improve diagnostic accuracy. Direct profiling of proteins in tissue sections using a technology termed MALDI mass spectrometry imaging (MALDI-MSI) provides a platform for revealing molecular details with possible diagnostic and prognostic implications. We have developed a strategy for the application of MALDI-MSI to the discovery of biomarkers that will improve the clinical management of prostate cancer. Our specific clinical targets are 1) detection of cancer, 2) detection of micrometastatic disease, and 3) detection of insignificant disease. We will present data from our analysis of approximately 100 tissue sections from stringently defined patient cohorts. We have also begun addressing the issues involved in positioning MALDI-MSI directly in the pathology workflow. Histology guided MS imaging is a promising strategy for identification of prostate cancer specific biomarkers that can be utilized to improve cancer diagnosis and most importantly for individualized and stratified patient risk for micrometastatic disease.
12:00-3:00 Pre-Conference Workshop* (*Separate Registration Required)
12:00-12:30 Occult Metastases Predict Recurrence Risk in Patients with pN0 Colorectal CancerScott A. Waldman, M.D., Ph.D., Chair, Department of Pharmacology and Experimental Therapeutics; Director, Gastrointestinal Malignancies Program, Kimmel Cancer Center, Thomas Jefferson University
Patients with stage I/II (pN0) colon cancer have a ~25% risk of disease recurrence reflecting under-diagnosis of micrometastases at staging. GCC, expressed selectively by intestinal cells and universally in colon tumors, is a marker of occult metastases in lymph nodes. Here, GCC was quantified by RT-PCR in lymph nodes collected from patients with pN0 colorectal cancer. Indeed, GCC qRT-PCR identified occult metastases that predicted the risk of disease recurrence in pN0 colorectal cancer. This prospective multicenter trial suggests that GCC is a prognostic and predictive molecular marker, identifying patients at increased risk for disease recurrence who could benefit from adjuvant chemotherapy.
12:30-1:00 Discovery and Validation of DNA Methylation-Based Biomarkers for Early Detection of Colorectal Cancer in PlasmaShannon Payne, Ph.D., Senior Scientist, Epigenomics, Inc.
Detection of colorectal cancer (CRC) at early stages has been shown to greatly decrease mortality from the disease. Availability of a blood-based test for CRC is expected to improve screening compliance in the general population. Through methylation-sensitive, restriction enzyme based marker discovery we identified a region of the Septin 9 (SEPT9) gene that is methylated in over 90% of colorectal cancer tissues with little or no methylation in normal colon tissue or other controls. Our process of biomarker development including real-time assay and preanalytics development, and successful application of the SEPT9 methylation biomarker to the specific detection of tumor DNA in multiple studies of plasma from CRC patients and controls will be described.
1:00-1:30 A Combinatorial Approach: The Use of MALDI-MS and Nano LC-MS for Glycan Biomarker DiscoveryTaufika Islam Williams, Ph.D., Research Assistant Professor, W.M. Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University
Epithelial ovarian cancer (EOC), the 5th leading cause of cancer deaths among women in the U.S., is characterized by poor survival statistics. Early-stage disease presents with few, if any, symptoms and diagnosis seldom occurs before metastasis has occurred. A 5-year survival rate of only 18% can be expected with late-stage disease detection. However, for the relatively few cases detected early, 88% of patients reach this survival mark. Insufficient sensitivity and specificity in established diagnostic modules is the primary reason why most patients are diagnosed in advanced disease, when prognosis is decidedly poor. We aim to apply MALDI-FT-ICR-MS and nano LC-MS in the investigation of carbohydrates chemically cleaved from glycoproteins in early-stage EOC plasma for cancer detection. Preliminary results have demonstrated complimentary glycan information provided by these two techniques.
1:30-2:00 Refreshment Break
2:00-2:30 Overcoming Potential Pitfalls in the Detection of Circulating Tumor Cells in Blood by RT-PCRLisa Roberts Rapp, Ph.D., Research Cell/Molecular Biologist, GPRD, Advanced Technologies Gene Expression Analaysis, Abbot Molecular, Inc.Detection of circulating tumor cells (CTC) in the blood of cancer patients may have prognostic and predictive significance. In an effort to detect CTC in breast cancer patients, RT-PCR for Cytokeratin 19 and HER-2 was employed. These two markers are relatively specific for epithelial cells and breast cancer cells, respectively. However, background expression of these ‘tumor specific markers’ in peripheral blood mononuclear cells (PBMC) may confound the analysis. We present a novel method to improve the specificity of the traditional method of detection of CTC by identifying the source of the background signals and reducing them using negative immunoselection.
2:30-3:00 Circulating Tumor DNA as a Biomarker of Colorectal CancerKerstin Schmidt, M.D., Ludwig Center for Cancer Genetics and Therapeutics, Johns Hopkins University School of MedicineMutant DNA fragments released by tumor cells into the circulation can be used to determine tumor burden and to monitor therapy response. In our study we longitudinally collected and analyzed 162 blood samples of 18 patients with colorectal cancer who underwent multimodality treatment with the sensitive BEAMing assay. The number of mutant DNA fragments was compared to radiologic findings and values of carcinoembryonic antigen during therapy. We could identify circulating tumor DNA in all patients and reliably monitor tumor burden during the course of therapy. We see this approach as a powerful and promising tool for monitoring therapy response and disease progression which could be easily extended to other cancer types in the future.
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