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Register by
February 16th and save up to $200!
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Tuesday, March 20
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8:00 Breakfast Workshop
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Sponsored by
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Increase Consistency from Sample to Ct
Ian Kavanagh, PhD. BSc., R&D Manager, ABgene,Thermo Fisher Scientific
There are many choices a researcher must make in order to establish a viable QRT-PCR procedure. We will discuss a comprehensive solution that minimizes variance and generates accurate data. This complete system will increase consistency from sample prep to validation with QRT-PCR.
8:45 Chair’s Remarks
8:50 PCR Bias in Methylation Independent PCR (MIP) Amplifications
Tomasz K. Wojdacz, M.Sc., Institute of Human Genetics, University of Aarhus, Denmark/Department of Molecular Pathology, Peter MacCallum Cancer
Centre, Denmark
Guidelines for MIP primer design advise avoidance of CpG dinucleotides in primer sequences or replacement of Cs within CpGs by a mismatched base. We have shown that a limited number of CpGs in the primer sequences may be necessary otherwise PCR bias towards the unmethylated template may lead to underestimation of the degree of methylation. Moreover, by manipulating the annealing temperature of the PCR amplification, we were able to control the efficiency of MIP primer binding to the methylated template and reverse PCR bias in favour of the methylated allele. Recently, the combination of the above strategy and High Resolution Melting technology (Methylation Sensitive High Resolution Melting, MS-HRM) allowed us to develop a panel of locus specific methylation assays capable of the detection of 0.1% methylated template in unmethylated background. Furthermore, we showed that MS-HRM can be designed to estimate the methylation level in a screened material. The overall parameters of MS-HRM make it the method of choice, readily applicable to both research and diagnostic settings.
9:10 CpG Island Methylator Phenotype (CIMP) in Colorectal Cancer Determined by Quantitative PCR (MethyLight)
Shuji Ogino, M.D., Ph.D., Assistant Professor of Pathology, Harvard Medical School, Department of Pathology, Brigham and Women’s Hospital, Department of Medical Oncology, Dana-Farber Cancer Institute
CpG island methylator phenotype (CIMP) has been established as a distinct phenotype in colorectal cancer. To discriminate biologically significant methylation from insignificant (low level) methylation, quantitative assays are necessary. Sodium bisulfite modification of genomic DNA and subsequent real-time TaqMan PCR (MethyLight) are robust with good precision and useful for paraffin-embedded tumor tissue. Quantitative PCR on carefully-selected CpG islands enables us to precisely diagnose CIMP status and should be utilized to assess effects of CIMP on clinical outcomes, patient survival and treatment efficacy.
9:40 Clinical and Molecular Responses in Lung Cancer Patients Receiving Chromatin Remodeling Agents
David S. Schrump, M.D., Head & Principal Investigator, Thoracic Oncology Section, Surgery Branch, Center for Cancer Research, National Cancer
Institute
Our published studies have demonstrated that 5 aza 2’ deoxycytidine (DAC) and Depsipeptide FK228 (DP) simultaneously de-repress cancer-testis and tumor suppressor genes, and mediate growth arrest and apoptosis in cultured lung cancer cells, but not normal bronchial epithelia. Presently, limited information is available regarding modulation of gene expression in primary lung cancers exposed to these chromatin remodeling agents. During the past several years, more than 80 patients with primary thoracic malignancies have received DAC, DP, or sequential DAC/DP infusions on protocols initiated in the Thoracic Oncology Section, Surgery Branch, NCI. Clinical toxicities, and response to therapy have been assessed by CTCAE and RECIST criteria, respectively. Plasma DAC and DP levels have been evaluated by LC-MS and HPLC techniques. Quantitative RT-PCR, methylation-specific-PCR, immunohistochemistry, and ELISA techniques have been used to assess a variety of molecular endpoints in pre-and post-treatment tumor biopsies and sera. Microarray techniques have been used to comprehensively examine gene expression profiles in laser-captured tumor cells from pre- and post treatment biopsies from 21 individuals receiving DAC, DP, or sequential DAC/DP infusions. Results of these arrays have been compared to data derived from analysis of laser-captured tumor cells and adjacent, histologically normal bronchial epithelia from 20 patients undergoing definitive lung cancer resections. Whereas no objective clinical responses have been observed, several patients have exhibited prolonged stabilization of disease following DAC, DP, or sequential DAC/DP infusions. Plasma DAC and DP concentrations have approximated threshold levels for gene induction and apoptosis in cultured lung cancer cells. Approximately 30% of patients receiving DAC or DP infusions have exhibited enhanced expression of NY-ESO-1, p16, p21, or acetylated core histones in tumor biopsies. Long-oligo array analyses have revealed complex, heterogeneous responses to DAC, DP, and DAC/DP in lung cancer cells, with an apparent shift of gene expression profiles toward those observed in histologically normal bronchial epithelia. Collectively, these data confirm that DNA demethylating agents and HDAC inhibitors can modulate gene expression in primary lung carcinomas. These preliminary observations support further evaluation of chromatin remodeling agents alone or in combination with treatment regimens, which either specifically target induced gene products, or modulate survival signaling in lung cancer cells.
10:10 Coffee Break, Poster and Exhibit Viewing
11:00 Application of Quantitative PCR Methodology to Gene Promoter Hypermethylation Analysis in Colon Cancers
Yuriko Mori, M.D., Ph.D., Assistant Professor of Medicine, Division of Gastroenterology, Department of Medicine, Johns Hopkins University
Assessment of gene promoter methylation status is a growing research field in cancer genetics. We employed quantitative PCR methodology for the rapid, sensitive, and quantitative detection of gene promoter hypermethylation in human cancers. This approach enabled us to identify several novel genes undergoing promoter hypermethylation in human primary colon cancers.
11:30 Panel Discussion with Morning Speakers
12:00-2:00 Luncheon Solutions Showcases
(Sponsorships Available)
12:00-12:30 BioMark™ System: A Breakthrough
Real-time qPCR System for HT Expression Profiling
Michael Lucero, Ph.D., Executive Vice President of Marketing, Fludigm
Corporation
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Sponsored by
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Diagnostic Development
2:00 Chair’s Remarks
2:05 Validating Molecular Diagnostics Methods For the Clinical Laboratory; Are We There Yet?
Dawn R. Maghakian, M.S., MP(ASCP), CLSp(MB), Research and Development, Molecular Diagnostics, Sacred Heart Medical Center
Since 1988, with the implementation of the Clinical Laboratory Improvement Amendments (`88 CLIA) by Congress, and the categorization of laboratory testing based on complexity by the Federal Food and Drug Administration (FDA), clinical laboratories must ensure that all test components and methods conform to federal regulations to meet accreditation goals. Today, with the increased availability of high-complexity molecular diagnostic test methods, validating these innovations to meet clinical regulatory needs presents significant challenges. Research scientists from both academic and corporate disciplines can expedite the process of moving novel genetic testing methods to the forefront of clinical medicine by understanding how test validation is accomplished in the clinical laboratory. The following topics will be discussed:
- The regulatory process in the clinical laboratory under `88 CLIA, an update
- Categories of test design and validation for the clinical laboratory: Evaluating RUO (Research Use Only), ASR (Analyte Specific Reagent) or “home brew” assays, and IVD or IVDMIA (In-vitro Diagnostic Multivariate Index Analytes) choices
- The validation process for molecular-based test methods in the clinical laboratory – key factors that will allow your discovery to transition into clinical use.
2:35 Verification of a Gene Expression Diagnostic for Cardiac Transplant Rejection
Robert Woodward, Ph.D., Director, R&D, XDx
Gene expression is increasingly considered valuable for informing on clinical pathology. These molecular pathology data have the promise to increase the accuracy of medical diagnoses. We have developed a gene expression test that informs on the status of cardiac transplant rejection by calculating a score from the expression levels of 11 pathology-related genes and 9 controls. This test was developed by comparison to known pathological outcomes and was verified in an independent patient population. The reproducibility of this real-time PCR test and the results in the prevalent population were also determined. The results of these verification and validation studies confirm that the test can be used to identify patients that are at low risk of experiencing rejection of their transplanted heart.
3:05 Refreshment Break (Last Chance for Poster and
Exhibit Viewing)
3:30 From Idea to IVD: Bringing Molecular Diagnostics to Market
Nicole Robinson, Ph.D., Principal Applications Scientist, Medical & Scientific Affairs, Roche Diagnostics Corporation
This talk will discuss the steps involved in bringing a product through the approval process starting with an overview of molecular diagnostics, explaining the different requirements for ASR, RUO, and IVD products, the five phases of development, a clinical trials overview, and conclusions.
4:15 Closing Panel of Experts
5:30 Close of Day |
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Clinical Trial Monitoring
2:00 Chair’s Remarks
2:05 Development of qRT-PCR Assays for Use in Monitoring Viremia in a Clinical Trial Setting
Linda Starr-Spires, Ph.D., Deputy Director, Nucleic Acid Methods Platform, Assay Development and Clinical Testing, Global Clinical Immunology Department, Sanofi Pasteur, Inc.
Assays intended for use in clinical trial monitoring are bound by restrictions unique to the setting. In developing qRT-PCR assays for such applications, it is necessary to consider not only typical development issues, but also take into account such things as workflow restrictions, increasing sample numbers, short turn around times, and regulatory requirements. Additionally, the assays are frequently directed at targets for which there are no commercial or compendial reagents available, requiring in-house manufacture and qualification of reagents by the development group. Approaches and solutions to these issues will be discussed.
2:35 Quantitative Real-time PCR Monitoring for Select Agent Disease Progression during Prophylaxis and Therapeutic Vaccine Efficacy Studies in Animal Models
Eric Lubert, Ph.D., Research Scientist, Molecular Biology/Toxicology, Battelle
Monitoring disease progression with quantitative real-time PCR (qPCR) has become a valuable tool for detecting and quantifying the presence of pathogen-specific nucleic acids within a wide variety of biological tissues. Our laboratory has developed qPCR assays for detecting and quantifying a wide range of select agents including highly-pathogenic avian influenza virus (H5N1), Monkeypox virus, Bacillus anthracis, and Yersinia pestis. Genetic material for these assays has been successfully isolated from several different animal species (i.e. mouse, rabbit, and nonhuman primate) and biological matrices (i.e. whole blood, peripheral blood mononuclear cells, saliva, and various organs/tissues). Reference plasmids possessing agent-specific gene targets and primer/probe sets were designed for use with the Taqman® reaction chemistry; optimization has allowed us to reach a sensitivity of ~10 copies. These qPCR assays were developed to enable monitoring of disease progression during prophylaxis and therapeutic efficacy studies, thereby providing vital information on tissue-specific agent distribution and titer. This information allows for defining the window of opportunity for therapeutic intervention and assessment of the protective levels of vaccines. This presentation will highlight our capabilities and approaches to developing, optimizing and eventual validation of qPCR assays. These assays will produce relevant, reliable and reproducible data for GLP animal efficacy studies performed to support licensure of medical products under the Animal Rule (21 CFR Part 601.90).
3:05 Refreshment Break (Last Chance for Poster and Exhibit Viewing)
3:30 Evaluation of a Real-Time QPCR Assay for HIV-1 Viral Load: A Monitoring Tool for Low-Level Replication in the Central Nervous System, Correlation with Antiretrovirals Penetration, and Implications for HIV-Associated Neurocognitive Disorders
Daniel R. McClernon, Ph.D., Principle Scientist, Clinical Virology, GlaxoSmithKline
Scott Letendre, M.D., Associate Professor of Medicine, HIV Neurobehavioral Research Center and Antiviral Research Center, University of California-San Diego
The real-time NucliSens EasyQ HIV-1 assay was developed for measurement of HIV-1 viral load in clinical specimens. Here we describe assay performance characteristics and its use as a possible tool for monitoring antiretroviral (ART) penetration and HIV neurocognitive impairment (HNCI). HNCI remains highly prevalent despite the use of combination antiretroviral therapy. Penetration of ART into the central nervous system can be low and this may allow ongoing replication and injury. Lower ART penetration was associated with detectable HIV RNA in cerebrospinal fluid (CSF) in a clinically relevant subgroup and in a small number of longitudinal specimens. These analyses indicate that a substantial proportion of individuals have HIV in CSF despite otherwise successful ART, which may explain the occurrence of HNCI in treated individuals.
4:15 Closing Panel of Experts
5:30 Close of Day |
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