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Here are the outstanding speakers you will hear at Planet xMAP USA 2010:

Keynote Speakers

Keynote Address - Wednesday, May 12
Dr. James D. Watson, Ph.D.
Nobel Prize-Winning Scientist and Co-Discoverer of the DNA Structure

Luminex Corporation
Presentation:  xMAP Technology Tutorial - The Fundamental Basics of Multiplexing

Sally Lewis, Ph.D.
Department Head Clinical Laboratory Sciences
Tarleton University

Presentation:  Lab Experience with xMAP Technology

John SantaLucia Jr., Ph.D.
Professor of Biochemistry
DNA Software, Inc.

Presentation:  Physical Principles and Software for Optimal Multiplex PCR Design

Abstract:
Optimal design of PCR reactions is a challenging problem, particularly as more complex formats such Multiplex PCR are introduced.  Secondary structural folding of DNA or RNA targets can inhibit hybridization causing false-negative PCR.  The presence of competing genomic DNA or contaminating organism genomic background can cause false-positive PCR.  Optimal Multiplex PCR design requires all probes/primers to be optimally sensitive and selective for their intended targets at the same reaction conditions.  Moreover, Multiplex PCR design must avoid destructive competing events such as target mis-hybridization, oligonucleotide cross-hybridization, and production of false amplicons.  This seminar will present some fundamental physical principles of DNA hybridization and folding and show how these principles apply to designing optimal Multiplex PCR reactions.  In addition, this seminar will highlight DNA Software’s novel Visual OMP™, ThermoBLAST™, and Modifieds™ software programs, which significantly improve Multiplex PCR performance.

DNA Software’s Visual OMP™, ThermoBLAST™, and Modifieds™ technologies contain advanced science and capabilities that are ideally suited for Multiplex PCR design.  Visual OMP™ incorporates its own dynamic programming algorithm for folding and secondary structure analysis.  In addition, Visual OMP™ uniquely contains an advanced algorithm for multi-state coupled equilibrium.  Accordingly, it folds and accounts for every possible interaction for each sequence in an experiment using actual solution conditions.  Moreover, Visual OMP™ produces cross-hybridization tables and graphs of Tm versus concentration for every possible sequence interaction.  Visual OMP™ also uniquely incorporates PCR additives (e.g. Luminex buffer, TMAC, and Betaine) plus many fluorophores and quenchers.  DNA Software uniquely offers ThermoBLAST™ and Modifieds™, which are two additional programs that significantly improve assay specificity and sensitivity.  ThermoBLAST™ was developed under a contract from the Department of Homeland Security for biodefense applications.  It combines the database capabilities of BLAST with the accurate thermodynamics of Visual OMP.  ThermoBLAST™ quickly and accurately scans DNA and RNA probes against large genome databases and discovers thermodynamically stable hybridizations and thereby eliminates assay false positives and off-target effects.  Modifieds™ designs assays, diagnostics, and therapeutics that incorporate modified nucleotides (e.g. LNA, PNA, Morpholino, DeoxyU, 5-methyl-C, Inosine, plus many more). 

Jochen Schwenk, Ph.D.
Proteomics School of Biotechnology
KTH Royal Institute of Technology

Presentation:  Towards a Next Generation Plasma Proteome Profiling

Abstract:
To pursue an affinity-based proteome analysis of human body fluids, (i) high-throughput technologies such as microarrays and (ii) large collections of validated protein-specific capture molecules are required. Such challenging approach is now initiated at the Human Protein Atlas resource (www.proteinatlas.org), which has been set-up to allow a systematic, antibody-based exploration of the human proteome (1). With more than 10,000 well-characterized affinity reagents being accessible for a presented next generation profiling effort an antibody suspension bead array has been developed for biomarker discovery in serum and plasma.

A microtiter plate-based workflow has been developed that combines optimized buffer, sample labeling and treatment (2) to now perform up to 384 tests in parallel on 384 samples per day. This workflow does consume only minute amounts of serum or plasma (3), and allows for a multiplexed and sensitive protein profiling. The system has now already been utilized for various biomarker discovery projects, and antibodies have been selected in both directed and undirected fashion to reveal promising differences in protein profile between patient groups (data unpublished). The overall results suggested that the application of this emerging suspension bead array workflow holds the great potential to enable a proteome-wide, undirected biomarker discovery in a larger number of samples.

Therefore, we have recently started to profile human serum and plasma in a systematic manner: In a discovery phase antibodies are selected, validated and applied to screen a multi-disease cohort composed of 384 patient samples. Then, interesting findings are further to be qualified in subsequent phase with dedicated single disease cohorts. In the nearer future technological advances will allow to perform up to 1.000.000 assays per week and as more long-term objective develop streamlined assays based on two antibodies (dual specificity) to further increase the sensitivity and to evaluate the true potential of the biomarkers.

Mark T. Esser, Ph.D.
Sr. Research Fellow, Operations
PPD Vaccines and Biologics Lab

Presentation: Multiplexed Assays for Multivalent Vaccines

Abstract:
Vaccine biomarker assays have been used historically to monitor disease incidence and prevalence, measure immune responses to natural infection or vaccination and to determine the duration of immunity.  This talk will discuss how multiplexed Luminex assays are being used to support clinical trials and post-licensure surveillance studies of licensed and new multivalent vaccines.  Specific examples of how PPD Vaccines and Biologics Lab is employing molecular, antibody and cytokine assays on the Luminex platform to support vaccine studies for influenza, human papillomavirus and diphtheria, tetanus and pertussis vaccines will be presented.

Takeshi Shimamura, Ph.D.
Instructor in Medicine
Dana-Farber Cancer Institute

Presentation:  Epithelial to Mesenchymal Transition Phenotype in Non-small Cell Lung Cancer Cell Lines Contributes to EGFR Inhibitor Resistance

Abstract:
Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, lead to significant tumor regressions in approximately 10% of non-small cell lung cancer (NSCLC) patients with EGFR activating mutations. However, up to 40% of NSCLC patients, majority of whom are EGFR wild-type, do not benefit from EGFR TKI therapy. A mesenchymal phenotype is well-correlated with insensitivity to EGFR TKIs and often found in NSCLC cell lines with wild-type EGFR, though the molecular mechanisms still remain elusive. Here we find both tyrosine phosphorylation of ErbB family and Met receptors are downregulated in mesenchymal-like tumor cells. Additionally, mesenchymal-like NCI-H1975CLR cells, which was grown resistant to irreversible EGFR TKI, exhibit aberrant insulin receptor (IR) and IGF-IR expression and activation of the PI3-K and Src signaling pathways. Pharmacological inhibition of IR and IGF-IR receptors reduced cell proliferation and compromised cell survival in mesenchymal-like but not parental NSCLC cell lines, suggesting the switching of the predominant receptor tyrosine kinases controlling cell proliferation and survival. Using multiplex bead based quantification of 15 growth factors, we found deregulation of the expression of EGFR ligands and TGFβ1 in the mesenchymal-like cells. Mesenchymal-like transition in NCI-H1975 cells was also achieved by exogenous addition of TGFβ1 and the cells showed many of the characteristics of epithelial to mesenchymal transition (EMT) including a loss of ErbB3 expression, decreased expression of E-cadherin, increased vimentin expression, and reduced sensitivity to irreversible EGFR TKI. Activation of IR/IGF-IR was observed in the TGFβ1 treated H1975 cells. The mesenchymal-like NSCLC cells showed increased migration and upregulation of intrinsic signaling pathways controlling cell adhesion. The findings of receptor tyrosine kinase switching and deregulation of intrinsic signaling pathways suggest investigation of new therapeutic targets in NSCLC with mesenchymal phenotype.

M. Sue Leffell, Ph.D., Diplomate, ABMLI, ABHI
Professor of Medicine and Director
Johns Hopkins Immunogenetics Laboratory

Presentation:  HLA/Gene Expression

Abstract:
Definition or “typing” of antigens and alleles of the human major histocompatibility gene complex, the HLA system, is performed as an integral component of solid organ and hematopoietic stem cell transplantation. As many transplant candidates become pre-sensitized to HLA antigens and form antibodies that become a barrier to transplantation, antibody detection and identification are also critical parts of histocompatibility evaluations. HLA typing is confounded by the number of HLA alleles: the HLA system is the single most polymorphic human genetic system with over 4000 defined alleles. Definition of the specificity of antibodies to HLA antigens becomes difficult when patients are highly sensitized with antibodies directed toward numerous antigens. The ability to incorporate multiple sets of oligonucleotide probes greatly facilitates HLA typing through a reverse hybridization technique. Similarly, coupling of purified HLA antigens to Luminex beads provides a highly specific and sensitive method for identification of HLA specific antibodies. Because of these features, Luminex®technology is one of the most widely used platforms in histocompatibility laboratories.

Laurie M. Clotilde-Bollinger, Ph.D.
Research Biologist
USDA Agricultural Research Service

Presentation:  Detection and Recovery of Shiga Toxins and Escherichia coli O157

Abstract:
Shiga toxin-producing Escherichia coli (STEC) are among the most costly foodborne pathogens.  In the United States, recent annual cost estimates for acute care ranged from $1 to $2 billion.  These were based on the assumption that E. coli O157:H7 led to 73,000 illnesses and 61 deaths each year.  The pathogenesis of these infections depends on the production of Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2).  Currently, there are no commercially available kits capable of detecting Stx1, Stx2, and O157 lipopolysaccharides (LPS) simultaneously.  As STEC strains producing Stx1, Stx2, or both toxins have often been linked to outbreaks of human illnesses and most of these are traced to consumption of E. coli O157-contaminated foods, this study focused on this specific STEC serotype as well as the main virulence factors.  Here, we developed a Luminex-based immunoassay to screen for Stx1, Stx2, and E. coli O157 LPS simultaneously in spiked foods.  Using minimal sample preparation, we were able to detect the three analytes simultaneously and our results showed the same specificity and sensitivity as ones obtained from testing pure STEC cultures.  Conventional sandwich ELISA using the same antibodies was not as sensitive.  Our newly developed Luminex-based immunoassay will serve as a milestone for developing a multiplex immunoassay for additional foodborne pathogens.

P. Scott White, Ph.D.
Acting Program Manager for Chem/Bio/Med Countermeasures
Los Alamos National Laboratory

Presentation:  Multiplex DNA/RNA-based Assays for Detection, Characterization, and Genotyping

Abstract:
Nucleic acid-based assays are ideal for pathogen detection and diagnostics, as well as for genotyping due to their high specificity, the ease with which they can be configured to detect almost any target, their requirement for minimal quantities of sample (high sensitivity), and their ability to be automated for high throughput applications. In addition, recent advances allow such assays to be configured in a multiplex format, enabling simultaneous screening for multiple genetic targets.  Examples of such applications include surveillance for multiple pathogens using multiple genomic targets for each, performing identification and genotyping on many sites simultaneously, or to perform extensive genetic characterization of samples of known identity.  There is a huge cost advantage to performing multiplex that allows even more targets to be interrogated, whereas cost is a common limiting factor for singleplex assays.
One challenge faced in developing assays to address such demanding applications is the lack of suitable and robust assays that perform well in multiplex. Although there are several analysis platforms capable of reading many different assay results, their utility is limited by the use of assays that do not perform well in a multiplex. In many cases, assays designed for singleplex formats are applied to multiplex analysis instruments with poor results. We have developed an assay, called Multiplex Oligonucleotide Ligation-PCR (MOL-PCR) that was designed for multiplex, and uses a flow cytometer for analysis. The assay uses generic or universal components where possible, which add much needed flexibility in design and reconfiguration, whereas specificity is conferred by careful nucleic acid target selection and probe design. Results of several Luminex-based MOL-PCR assays that address a diverse collection of detection and surveillance applications will be presented.

Gary Procop, M.D., M.S.
Chairman of the Department of Clincial Pathology
Cleveland Clinic Foundation

Presentation:  Molecular Infectious Disease

Christine Ginocchio, Ph.D., M.T. (A.S.C.P.)
Director of Microbiology and Molecular Diagnostics
North Shore LIJ Health System Laboratories

Presentation:  Molecular Infectious Disease xTAG Respiratory Viral Panel

Jean Louis Merlin, PharmD, Ph. D.
Alexis Vautrin Cancer Center

Presentation:  Phosphoprotein Array Assay for Characterization of Human Tyrosine Kinase Receptors Downstream Signaling Functionality as Response Predictive Marker to Targeted Therapy in Translational Oncology Research Programs

Abstract:The functionality of human epidermal growth factor receptors (HER) downstream signaling kinases have major consequences on tumor response to anti-HER targeted therapy. In our research project, Luminex® phosphoprotein array (PA) was used to analyze the expression of p-MEK, p-ERK1/2, p-P38MAPK, p-AKT, p-GSK3, p-P70S6K in cell lines (preclinical models) and in frozen biopsies taken at diagnosis from breast, head and neck and colorectal cancer patients.

In preclinical transgenic cells, PA was found to be very useful in understanding the role of key-determinants of the cellular response to anti-HER directed drugs or stategies (Cancer Gene Ther, 2009). In patients, using optimized standard operating procedures (freezing delay, specimen quality, protein extraction), PA was cross-validated with western blot analysis in 49 breast cancer specimens and the two methods showed clinical comparability (Clin Chem, 2009). In all tumor types, great variations (up to several-hundred folds) in phosphoprotein expression were observed among the specimens revealing high-range inter-individual variations. In metastatic colorectal cancer, PA results were confronted to KRAS mutation, used today as response predictive marker to anti-EGFR monoclonal antibodies. KRAS and p-MEK were identified as two independent prognostic markers of patients treated by cetuximab. In wild type KRAS tumors, p-MEK and p-P70S6K overexpression were found to be associated with significant lower progression-free survival showing that PA could be used for clinical response prediction (Int J Cancer, 2010).

As a whole, our results validate the use of PA for single-step analysis of signaling pathways functionality in translational oncology research programs. In either preclinical or clinical research programs, PA is a potent tool for evaluation and validation of molecular determinants / clinical response predictive markers of cellular / tumor response to anti-HER directed targeted therapy.

Matthew Myles, D.V.M., Ph.D., DACLAM
Research Animal Diagnostic Laboratory
Veterinary Pathology
College of Veterinary Medicine
University of Missouri

Presentation:  Immunodiagnostics-Serology Diagnostics

FLEXMAP 3D Lunch Session - Thursday, May 13