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Digital PCR Conference: Technologies and Tools for Precision Diagnostics - Day 2



Day 1 | Day 2 | Day 3 | Download Brochure 

TUESDAY, OCTOBER 7

7:45 am Problem Solving Breakout Discussions with Continental Breakfast

TABLE 1: Sources of Error when Measuring Nucleic Acids Using Digital PCR?

Moderator: Jim Huggett, B.Sc. (Hons), Ph.D., Science Leader, Nucleic Acid Metrology, Molecular & Cell Biology, LGC

  • Variance
  • Bias
  • Absolute quantification/rare variant detection
  • Need for controls/callibration

TABLE 2: Fractionated Cell-Free DNA: The Somewhat Other Moiety

Moderator: Ekkehard Schütz, M.D., Ph.D., CTO, Senior Vice President, Research, Chronix Biomedical, Inc.

  • Genomic DNA vs cfDNA
  • Amplifyable fraction
  • Bias control
  • Measured vs. true values

TABLE 3: Digital PCR Data Analysis

Moderator: Jo Vandesompele, Ph.D., CEO, Biogazelle; Professor, Ghent University

  • What are your current challenges?
  • Where do you see room for improvement?
  • Do the digital MIQE help?
  • What is the best advice you can offer?

dPCR FOR PATIENT MONITORING AND STRATIFICATION  

8:55 Chairperson’s Opening Remarks

Chris Karlovich, Ph.D., Clovis Oncology

 

9:00 Digital PCR for Patient Monitoring and Stratification in Clinical Trials

Reinhold Pollner, Ph.D., Director, Clinical Trial Assay Development, Genoptix Medical Laboratory, a Novartis Company

My presentation will focus on how digital PCR is used in the validation of clinical trial assays with respect to patient monitoring and stratification in a CLIA/CAP laboratory setting. A variety of different applications of digital PCR will be discussed ranging from the determination of DNA copy numbers in FFPE samples, absolute quantitation of a transgene in whole blood and bone marrow samples, identification of a fusion gene at the mRNA level in FFPE specimens, and rare allele detection using circulating tumor DNA.

9:30 Evaluation of EGFR Mutations in Plasma from NSCLC Patients: Utility in Managing Patients on TKI Therapy

Chris Karlovich, Ph.D., Principal Scientist, Molecular Diagnostics, Clovis Oncology

We are utilizing blood-based molecular testing in patients who have become resistant to first generation tyrosine kinase inhibitors with the goal of enabling subsequent therapy without need for repeat lung biopsy. The utility of plasma-based EGFR mutational analysis will be described in the context of CO-1686, a novel third-generation TKI that selectively inhibits the EGFR activating and T790M resistance mutations in NSCLC patients.

10:00 Serial Monitoring of EGFR Mutations in Plasma and Matched Tissue from EGFR Mutant Non-Small Cell Lung Cancer Patients on Erlotinib

Cloud P. Paweletz, Ph.D., Head, Translational Research Laboratory; Biomarker Lead, Belfer Institute for Applied Cancer Science, Dana Farber Cancer Institute

We report on the detection, quantification and monitoring of EGFR mutations by droplet digital PCR in cfDNA on a prospective clinical trial of EGFR-mutant NSCLC patients on erlotinib. EGFR L858R, Del(19) and T790M were serially measured in over 300 samples from 40 lung cancer patients treated on erlotinib. Complete plasma response was seen in 8 cases; in 6, rising levels of inhibitor sensitizing EGFR Del(19) and L858R and acquired EGFR T790M mutations were detected up to 4 months prior to objective radiographic progression. Our results strongly suggest that cfDNA genotyping has clinical utility in the molecular assessment of patients at diagnosis, and providing molecular understanding of patient’s tumor evolution.

10:30 Coffee Break with Exhibit and Poster Viewing

 

MINORITY TARGET DETECTION  

11:15 Digital PCR for Detecting Rare Genomic Alterations which Enhances Sensitivity in Cell-Free DNA

Rachel Tam, Senior Research Associate, Clinical Assay Development, Oncology Biomarker Development, Genentech, Inc.

11:45 Applications of Digital PCR in Oncology: Detecting Residual Disease in Plasma of Early Stage Breast Cancer Patients and Archival Tissue Studies Using Droplet Digital PCR

Rory L. Cochran, Ph.D., Medical Student, UC San Diego, School of Medicine

This presentation will focus on the rapidly emerging applications of digital PCR in both clinical oncology and oncology research. Recent work in early stage breast cancer patients demonstrating the remarkable sensitivity and specificity of using plasma tumor DNA (ptDNA) will be discussed. In addition, advances in oncology research using archival tissue derived DNA will be covered. Droplet digital PCR improves upon previous methods and overcomes challenges for establishing accurate DNA copy number changes, quantifying allelic fractional abundance and assessing loss-of-heterozygosity in formalin fixed DNA samples.

12:15 pm Luncheon Presentation (Sponsorship Opportunity Available) or Lunch on Your Own

12:45 Session Break

 

COPY NUMBER VARIATION  

1:25 Chairperson’s Remarks

Douglas Sanders, Ph.D., Novartis

1:30 CYP2D6 Allele Specific Copy Number Analysis Using TaqMan® SNP Genotyping Assays and Digital PCR

Russell_IainIain Russell, Ph.D., Senior Product Manager, Genetic Analysis, Thermo Fisher Scientific

The major drug metabolizing enzyme, CYP2D6, is encoded by a highly polymorphic gene. For samples that carry CYP2D6 duplications and are heterozygous for key SNPs, the specific allele that is duplicated cannot always be identified. To address this issue, we developed a method to detect allele-specific copy number by digital PCR using the QuantStudio™ 3D Digital PCR System. In this session, we demonstrate that this simple and effective method can facilitate accurate CYP2D6 allele genotyping and better prediction of a drug metabolizing phenotype.

2:00 Development of Configurable Genotyping Assays for Single Color Digital PCR in Cancer

Hanlee P. Ji, M.D., Assistant Professor, Medicine, Division of Oncology, Stanford University School of Medicine

We have developed several novel digital PCR assays that can used for copy number or point mutation discrimination from a variety of clinical samples. Even in the context of dilution mixtures, we are able to achieve very high sensitivity and specificity. These assays rely on single color assays and avoid Taq probe development.

2:30 Evaluation of Genetic Stability of HSV Transgenes in a Constructed Vero Cell Line

Ali Azizi, Ph.D., Scientist, Adjunct Professor, Microbiology and Virology, Analytical Research and Development, sanofi pasteur

The genetic stability of transgenes is a critical characteristic used to assess constructed cell lines used for vaccine production. To assess the genetic stability of the transgenes in a constructed Vero cell line, a digital PCR was developed. The developed assay was used to monitor the stability of the transgenes by comparison of copy numbers in the master cell bank with their copy numbers in the extended cell bank.

3:00 Refreshment Break with Exhibit and Poster Viewing

3:45 Detection and Quantification of Alternative Splice Forms and mRNA Processing Intermediates using Droplet Digital PCR

Dianna Maar, Ph.D., Senior Scientist, Applications, Bio-Rad Laboratories

Alternative splicing and the regulation of RNA processing play a pivotal role in the expression of genetic information and in establishing a cell’s phenotype. Mutations in target transcripts and in RNA processing factors can lead to both germline diseases and cancer and represent potential diagnostic and therapeutic targets. This talk will demonstrate the utility of droplet digital PCR for unraveling the complexities of this intricate network.

4:15 Molecular Methods for Biological Quality Control: Danger Tool or Perfect Solution?

Julie R. Murrell, Ph.D., Program Manager for Collaborations, R&D Manager - Stem Cell Biology Lab, Stem Cell Bioprocessing Group, EMD Millipore

The concept of using molecular biology and PCR-based testing methods to check the viral safety of biotech processes is quite appealing and novel: the same PCR application could be considered misleading if it provides false positive results, or the fastest method to find out everything you need to know about your sample. Facility design, GMP compliance, highly trained personnel and next generation technologies really make the difference in molecular biology method development and validation for biopharmaceuticals quality control. Next generation sequencing approaches and case studies in viral and mycoplasma detection, automation, and more GMP applications to secure manufacturing facilities and shorten testing timelines will be presented.

4:45 Statistical Modeling Techniques for Setting Digital PCR-Based Quality Control Specifications

Douglas Sanders, Ph.D., Senior Scientist, Companion Diagnostics, Novartis

Digital droplet PCR technology is a precision tool for quantitative bioanalytics. Setting equally precise assay parameters and specifications requires an accurate mathematical model of the assay performance characteristics. This presentation explores methods used to develop quality control assays and set assay specifications using Six Sigma techniques.

5:15 Close of Day; Dinner Short Course Registration

6:00 - 8:00 Dinner Short Course: Single-Cell Genomic Analysis*

Daniel T. Chiu, Ph.D., A. Bruce Montgomery Professor, Chemistry; Endowed Professor, Analytical Chemistry; Professor, Bioengineering, University of Washington, Seattle

Jason Kreutz, Ph.D., Senior Scientist, Chemistry, University of Washington, Seattle

Amy Paguirigan, Ph.D., Staff Scientist, Fred Hutchinson Cancer Research Center

* Separate registration required

 

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