Here are the outstanding speakers you will hear at Planet xMAP USA 2008:

Carlo Croce, M.D. - Wednesday, March 5
John W. Wolfe Chair in Human Cancer Genetics, Professor/Chair, Molecular Virology, Immunology and Medical Genetics, Director, Human Cancer Genetics Program at The Ohio State University, Comprehensive Cancer Center

Presentation:  MicroRNA-29 family reverts aberrant methylation in lung cancer by targeting DNA methyltransferases 3A and 3B

Abstract:  MicroRNAs (miRNAs) are small, noncoding RNAs that regulate expression of many genes.  Recent studies suggest roles of miRNAs in carcinogenesis.  We and others have shown that expression
profiles of miRNAs are different in lung cancer vs. normal lung, although the significance of this aberrant expression is poorly understood.  Among the reported down-regulated miRNAs in lung cancer, the miRNA (miR)-29 family (29a, 29b, and 29c) has intriguing complementarities to the 3’-UTRs of DNA methyltransferase (DNMT)3A and -3B (de novo methyltransferases), two key enzymes involved in DNA methylation, that are frequently up-regulated in lung cancer and associated with poor prognosis.  We investigated whether miR-29S could target DNMT3A and –B and whether restoration of miR-29s could normalize aberrant patterns of methylation in non-small-cell lung cancer. Here we show that expression of miR-29s is inversely correlated to DNMT3A and –B in lung cancer tissues, and that miR-29s directly target both DNMT3A and –B.  The enforced expression of miR-29s in lung cancer cell lines restores normal patterns of DNA methylation, induces reexpression of methylation-silenced tumor suppressor genes, such as FHIT and WWOX, and inhibits tumorigenicity in vitro and in vivo.  These findings support a role of miR-29s in epigenetic normalization of NSCLC, providing a rationale for the development of miRNA-based strategies for the treatment of lung cancer.

Carlo Croce, M.D.
Dr. Croce is world-renowned for his contributions involving the genes and genetic mechanisms implicated in the pathogenesis of human cancer.  During the course of his career, he discovered the juxtaposition of the human immunoglobulin genes to the MYC oncogene, the deregulation of MYC in Burkitt lymphoma, the ALL1 gene involved in acute leukemias, the TLC1 gene associated with T-cell leukemias, and cloned and characterized the Bcl2 gene involved in follicular lymphoma.  Dr. Croce has also uncovered the early events involved in the pathogenesis of lung, nasopharyngeal, head and neck, esophageal, gastro-intestinal and breast cancers.  Recently he discovered the involvement of miR genes in human cancer.

His discoveries have led to revolutionary innovations in the development of novel and successful approaches to cancer prevention, diagnosis, monitoring and treatment, based on gene-target discovery, verification and rational drug development.

Christine Falk, Ph.D.
National Center for Tumor Diseases (NCT), German Research Center, DKFZ and the Institute for Immunology in Heidelberg, Immune Monitoring Unit
Group Leader

Presentation:  The Multiplex Technology in Immunology: Cytokines, Phosphokinases and HLA/KIR Typing

Abstract: Immune cells, in general, are regulated by receptor-mediated signaling evens that are transduced into their individual effector functions like cytokine secretion, proliferation or cytotoxicity, for instance. The bead-based Luminex Technology opens the possibility to analyze these signalling events, their functional consequences (cytokine secretion) as well as the genetic background on a high-resolution level – with minimal time (and appropriate) financial requests. We analyzed these features with human Natural Killer (NK) cells because recent evidence suggests that NK cells may play an important role in halploidentical bone marrow / stem transplantation (BMT/SCT) as well as in autoimmunity.

A sophisticated system of activating and inhibitory receptors regulates NK effector functions. Most NK receptors bind to HLA class I molecules, HLA-C in particular. Two receptors of the killer immunoglobulin-like receptor family (KIR) discriminate between two HLA-C allele families which are defined by their amino acid compositions in position 77 and 80 of the a1-helical domain. Therefore, it is mandatory to define all HLA-C alleles of BMT/SCT donors and leukemia patients at the molecular level. In addition, the KIR genes are organized in a haplotype system with considerable polymorphism with respect to allelic variants of single receptors and the presence or absence of specific genes. Therefore, high resolution HLA-C typing and KIR typing was performed. . We are currently analyzing the HLA-C/KIR genes of 80 donor/recipient combinations following haploidentical BMT/SCT.

To evaluate the potential of the Multiplex Technology for the analysis of NK cells at the functional level, we performed extended cytokine analysis and analysis of total and phorphorylated cell signaling molecules using miniaturized and parallelized sandwich immunoassays. Cytokine secretion patterns of several NK lines and alloreactive T cell clones were detected with respect to inducible or constitutive production of IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IFN-g, TNF-a and MIP-1ß. Stimulation of activating NK receptors resulted in different cytokine patterns in various NK lines indicating that at least some NK cells possess an individual cytokine repertoire. The underlying signalling events were analyzed by semi-quantitative phosphokinase detections of selected MAP and src kinases. These results were compared with standard western blots demonstrating an excellent correlation of the two methods. In conclusion, the combination of functional and genetic analyses allows a more comprehensive understanding of the complicated NK cell regulation and this information is of fundamental importance in order to define the relevance of NK cells for hematopoietic malignancies and autoimmunity.

Christine Falk, Ph.D.
Christine Falk (Ph.D., Immunology, Heidelberg, Germany) is an immunologist and leads the Immune Monitoring Unit at the National Center for Tumor Diseases (NCT) at the German Research Center, DKFZ and the Institute for Immunology in Heidelberg. Her major interest is the recognition of tumor cells by specific cytotoxic T cells (CTL) and Natural Killer (NK) cells and the quantification of these immune responses in patients receiving immune therapy. She has more than 30 publications and patents in immune regulation, immune response to viruses or tumor cells, regulation of ligand expression for NK and T cells by cytokines and small molecules and the genetics of the Human Leukocyte Antigen (HLA) and the killer immunoglobuline-like receptors in bone marrow and stem cell transplantation. She is PI in various national and international grants and member of the review committee for international journals.

Presentation:  Application of Multiplex Bead-based assays in Preclinical and Clinical Cancer Research

Abstract:  A major focus of our biomarker efforts is to develop a broad, comprehensive blood-based biomarker platform for assessing the activity of targeted agents and developing profiles for predicting therapeutic sensitivity or resistance. Such a platform would offer the potential for non-invasive evaluation of patients over time and lends itself to high thoughput analyses. The utilization and development of the multiplex assay have increased the potential usefulness and providing a data-rich environment. Preliminary results from various experiments and studies have fueled further development of this platform from preclinical studies in cell-based studies to animal and then to pilot clinical studies, and finally to application to randomized phase II/III clinical trials in cancer therapy; specifically in lung and head/neck cancers. Our current platform contains two main components: a cytokine/angiogenic factor (HCF) serum/plasma profile, and cellular analyses including CECs/CEPs and other target-bearing PBMCs. Current plan is to broaden the profile to include additional factors, particularly members of the IGF axis (IGF-1, IGF-2, IGF-BP3) and additional EGFR family ligands, and apply it to various study in both preclinical and clinical platforms. One current application have been to implement these biomarkers analysis into our therapeutic area targeting antiangiogenesis; these biomarkers include plasma VEGF, sVEGFR-2, sE-selectin proangiogenic cytokines directly involved in the relevant targeted pathways (VEGF, EGF, TGF-alpha, IGF-I, IGF-II, IGF-BP3), or that may contribute to therapeutic resistance by promoting the proliferation and survival of tumor endothelial cells even in the presence of VEGF blockade (i.e. bFGF), markers of hypoxia, or chemokines or interleukins, many of which are thought to play a direct or indirect role in angiogenesis mediated by the inflammatory system.

Hai Tran, PharmD
Hai T. Tran, PharmD, is assistant professor of cancer medicine and pharmacology with a joint appointment with Divisions of Cancer Medicine and Pharmacy at the University of Texas MD Anderson Cancer Center (MDACC) in Houston. After earning his BS degree from Temple University, Philadelphia, Pennsylvania, he went on to earn his doctor of pharmacy degree from the University of Kentucky in Lexington. His postgraduate training includes a clinical pharmacy residency in oncology and a clinical pharmacology research fellowship, both at UTMDACC. Dr. Tran has been active in research during the course of his career, having served as principal or co-principal investigator on a number of pharmaceutical company-sponsored trials. His major research focuses are new drug development, pharmacokinetics/pharmacodynamics models, and blood-based biomarkers. He is a member of such prestigious professional organizations as the American Societies of Clinical Oncology and Hematology and the American Association for Cancer Research. He authored/co-author over 25 journal articles. Dr. Tran's most recent article was published in Clinical Cancer Research, Nature's Medicine, Cancer Chemotherapy and Pharmacology and Biology of Blood and Marrow Transplantation.

Luis Giavedoni, Ph.D.
Southwest Foundation for Biomedical Research 
Scientist, Director SNPRC Flow Cytometry Core Facility, Interim Associate Director for Research
SNPRC Department of Virology and Immunology

Presentation:  Use of the Luminex xMAP Technology for Validation of Nonhuman Primates as Best Animal Models for Human Diseases

Abstract:  Nonhuman primates (NHP) are the preferred animal models for pre-clinical research because they approximate humans in physiology and genetics more closely than any other animal.  Identification of immune mediators such as cytokines in NHP is crucial for the understanding of complex physiological and pathological mechanisms that occur in these species, and to demonstrate whether these mechanisms function similarly in humans.  We have a developed a panel of reagents that allow for the simultaneous identification of 32 cytokines in several NHP species.  Additionally, we have also applied the Luminex technology to the detection of monkey antibodies directed to several components of a viral particle; this technique can be applied to the evaluation of vaccine immunogenicity and efficacy in this relevant animal model.

Luis Giavedoni, Ph.D.
Dr. Giavedoni graduated as a Biochemist in 1982 from the National University of Litoral, in Santa Fe, Argentina. In 1986 he received a Doctorate in Biochemistry from the National University of Buenos Aires, also in Argentina.  In 1989 he moved to the University of California at Davis, where he spent 7 years, first as a post-doc and later as a Research Assistant Professor.  Since 1996 he has been at the Southwest Foundation for Biomedical Research (SFBR), in which he currently has the position of Scientist in the Department of Virology and Immunology, and Interim Associate Director for Research at the Southwest National Primate Research Center (SNPRC); Dr. Giavedoni is also Director of the Flow Cytometry Core Facility of the SNPRC.  Dr. Giavedoni’s expertise is the in the field of vaccine development and nonhuman primate immunology, particularly in the simian immunodeficiency virus/rhesus macaque animal model of AIDS.

Kimberly Stegmaier, M.D.
Dana-Farber Cancer Institute and Children’s Hospital Boston
Assistant Professor of Pediatrics,
Broad Institute of Harvard University and Massachusetts Institute of Technology
Associate Member

Presentation:  Signature-based Approaches to Small Molecule Library Screening

Abstract:  Despite progress in understanding the pathogenesis of malignancies, translation of scientific knowledge to clinical benefit remains slow. The genomic revolution has enabled a more granular approach to disease characterization, yet therapy development still lags behind. Traditional approaches to small molecule discovery have largely focused on simple phenotype-based (i.e. cell-death assays) or target-based screens. Each of these has played an important role in the drug discovery process, but each also has its limitations. As such, a host of challenges continue to impede compound discovery efforts:

1. Current methods of small molecule library screening have been limited.
2. Cell-free biochemical assays do not recapitulate the complexity within a cell.
3. Many potential targets have been considered pharmacologically intractable.
4. Often the target of disease pathogenesis is not known.

A more systems-based approach to small molecule library screening has the potential to overcome these challenges. With the sequencing of the human genome and the advent of genome-wide expression profiling, alternative approaches to chemical biology become feasible. Several new tactics have been explored, each using gene expression signatures to facilitate compound discovery. Our initial efforts focused on the use of multi-gene signatures as proxies for highly complex cellular networks in a small molecule library screen. In Gene Expression-based High-throughput Screening (GE-HTS), gene expression signatures of the biological states of interest (i.e. “state A” versus “state B”) are first defined using genome-wide expression profiling. Next, ligation-mediated amplification with fluorescent-bead based detection of amplicons (Luminex Platform) is used for the high-throughput, low-cost measurement of the signature. Then, a small molecule library is screened for compounds that induce a change from the “state A” to the “state B” signature. Unlike traditional phenotype-based screens, GE-HTS does not require development of specialized assays. The signature definition, amplification, and detection are generic. Furthermore, a priori knowledge of a target is not needed because the signature serves as a surrogate for the biological state in question. Our first proof of principle experiments successfully applied GE-HTS to the identification of compounds that induce differentiation in acute myeloid leukemia. Our second published screen applied GE-HTS to modulating a pharmacologically intractable target. In this work, we identified compounds that modulate the oncoprotein EWS/FLI in Ewing sarcoma. This oncoprotein fuses the transactivating domain of EWS to the ETS transcription factor FLI. Both screens have resulted in ongoing clinical trials. These preliminary results suggest that GE-HTS could be a complementary approach to traditional small molecule library screening that can be adopted widely. Signature-based approaches should enable systematic exploration of diseases and biological processes not previously studied and the identification of new therapeutic leads.

Kimberly Stegmaier, M.D.
Kimberly Stegmaier, M.D., is an Assistant Professor of Pediatrics at Harvard Medical School, Investigator at Dana-Farber Cancer Institute (DFCI), and an Associate Member of the Broad Institute of Harvard and MIT. She also attends in Pediatric Oncology at DFCI and Children’s Hospital Boston. Dr. Stegmaier received her undergraduate degree from Duke University, medical degree from Harvard Medical School, and trained in Pediatrics and Pediatric Hematology/Oncology at Children’s Hospital Boston and DFCI.

Her post-doctoral research, mentored by Dr. Todd Golub, developed a new signature-based approach to small molecule discovery, Gene Expression-based High-throughput Screening (GE-HTS), in which gene expression signatures serve as surrogates for complex biological states.

Christine Robinson, Ph.D.
Denver Children's Hospital
Coordinator of Research, Development and Outcomes, Microbiology/Virology Laboratory

Presentation:  The xTAG Respiratory Viral Panel

Abstract:  Respiratory viruses are a major cause of lower respiratory tract illness, especially in children. Timely diagnosis of respiratory viral disease has great potential to direct antiviral therapy, reduce management costs, improve antibiotic stewardship, and contribute to infection control, epidemiology, and surveillance studies. This presentation describes the Luminex Diagnostics xTAG™ Respiratory Viral Panel, a sensitive, same-day assay for the simultaneous detection of the predominant respiratory tract viruses, including RSV (A, B), influenza A (H1, H3, H5), influenza B, human metapneumovirus, adenovirus, parainfluenza viruses 1, 2, 3 and 4, enterovirus/rhinovirus, and coronaviruses OC43, 229E, Nl63, and HKU1.

Christine Robinson, Ph.D.
Dr. Robinson established the Virology Laboratory at The Denver Children’s Hospital (now in Aurora CO), which is ranked as one of the top 5 best children’s hospitals in the US. She also coordinates the Research, Development and Outcomes Program for the Microbiology and Virology Laboratories, advises the Molecular Diagnostic Laboratory, is active in the Pan American Society for Rapid Viral Diagnosis, and teaches extensively. The focus of her work is improvement of patient care by implementation and appropriate utilization of viral diagnostic technology. She received her BA in Biology from the University of California, a Masters Degree in Microbiology from Pennsylvania State University, a Ph.D. in Pathology from the University of Colorado, and did postdoctoral work in Molecular Virology at the University of Connecticut Health She has authored many manuscripts and several book chapters in her field.  

Kristen Keeling Reynolds, Ph.D.
PGxL Laboratories
Director of Laboratory Operations
Assistant Clinical Professor, Dept. Pathology and Laboratory Medicine, University of Louisville School of Medicine

Presentation:  New Paradigms in Personalized Medicine: Predictive Models in Warfarin Pharmacogenetics

Abstract:  Pharmacogenetic testing has the potential to revolutionize the practice of medicine; however, the direct application of clinical pharmacogenetics to the creation of individualized dosing strategies is dependent on how well genotyping results convey critical information.  Currently, one of the most advanced applications of pharmacogenetics is genotyping to predict hypersensitivity to the anticoagulant Coumadin (warfarin). The development of comprehensive, individualized predictive models for warfarin dose which integrate all of the relevant genetic and physical factors can be used to translate the results of pharmacogenetic testing into actionable clinical application.  Development of the warfarin genotyping technology from Luminex together with a comparison of these dose prediction models will be discussed.

Kristen Keeling Reynolds, Ph.D.
Dr. Reynolds is the Director of Laboratory Operations for the Pharmacogenetics Diagnostic Laboratory, LLC (PGXL Laboratories) in Louisville, KY, the first CLIA-certified laboratory to exclusively offer clinical pharmacogenetics testing in the United States.  She is also an Assistant Clinical Professor in the Department of Pathology and Laboratory Medicine, University of Louisville School of Medicine.  Following completion of postdoctoral fellowships in Clinical Chemistry and Advanced Pharmacogenetics at the University of Louisville, Dr. Reynolds has specialized in the discipline of clinical pharmacogenetics with emphasis on the technical and regulatory management of a clinical pharmacogenetics laboratory as well as development and implementation of new technology and applications for such a laboratory. 

Dr. Reynolds is actively involved in numerous professional societies, including the American Association for Clinical Chemistry (AACC), Association for Molecular Pathology (AMP), Clinical Ligand Assay Society (CLAS), and International Association for Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT).

Gary Procop, M.D.
Department Chairman, Clinical Pathology
Cleveland Clinic Foundation
 
Presentation:  The Detection and Differentiation of the Causes of Fungemia using Broad-Range PCR and Luminex xMAP® technology

Abstract:  Introduction:  Fungal infections are a major cause of morbidity and mortality in immunocompromised and severely ill patients. Fungemia is caused predominantly by one of four Candida species, C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis. Prompt, appropriate identification of the infecting species guides antifungal therapy. 

Materials and methods:  We are investigating the feasibility of using a broad-range (i.e. pan-fungal) PCR that targets the D2 region of the 28S rRNA gene in conjunction with a Luminex® xMAP® technology-based suspension array hybridization assay to more rapid identify Candida species. 21Candida parapsilosis, 21Candida glabrata, 18Candida tropicalis, and 12 Candida albicans clinical isolates were tested in the first phase of this study, as well as one ATCC strain of each organism. After PCR, hybridization was performed with multiplexed suspension array containing a mixture of spectrally distinct microsphere sets covalently coupled to species-specific capture probes. Amplicons were biotinylated to allow labeling with streptavidin-phycoerythrin (SAPE) reporter. Detection was done on a Luminex flow analyzer, where the microspheres were classified by their unique spectral address using a 635 nm laser and bound amplicons quantified using a 532 nm laser. Amplification was determined through probe hybridization and/or gel electrophoresis.

Results and conclusion will also be discussed.

Gary Procop, M.D.
Gary W. Procop, MD, MS is Chairman of the Department of Clinical Pathology at the Cleveland Clinic, and Medical Director of the Molecular Microbiology, Mycology, and Parasitology laboratories.  He completed Anatomic and Clinical Pathology training at Duke University Medical Center and a Clinical Microbiology Fellowship at the Mayo Clinic.  He is a diplomate of the American Board of Pathology in Anatomic and Clinical Pathology, and Medical Microbiology. He is a Fellow of the American Academy of Microbiology, the College of American Pathologists, and the American Society for Clinical Pathology.  He has given more than 300 scientific presentations, and has more than 100 publications and 23 chapters to his credit.  His primary interests are the practical applications of molecular diagnostic methods for the diagnosis and treatment of infections; infectious disease pathology; mycology and parasitology.