Digital PCR

Digital PCR continues to be an important addition in diagnostic labs, with more and more groups adding this technology to their catalogue of detection and quantification tools. Cambridge Healthtech Institute’s Fifth Annual Digital PCR symposium will discuss technological aspects of dPCR tools and technologies, including validation and accuracy for dPCR data, how dPCR integrates with NGS, and current and future applications of the technology, from single-cell analysis and diagnostics to environmental monitoring. Hear from researchers, test developers, and platform users on their experiences with and the limitations and benefits of dPCR, with a special focus on clinical applications. Click here to see who's attending the symposium.


SC2: Digital PCR: A Technology Primer

*Separate registration required


8:00 am Registration & Morning Coffee


8:25 Chairperson’s Opening Remarks

Hatim Husain, M.D., Assistant Professor, Medicine, Hematology and Oncology, University of California, San Diego

8:30 DNA Methylation Analysis Using Droplet Digital PCR

Ming_YuMing Yu, Ph. D., Staff Scientist, Clinical Research, Fred Hutchinson Cancer Center

Droplet digital (ddPCR) is a recent advance in PCR technology that enables the precise detection and absolute quantification of nucleic acid target sequences that has a range of applications for both research and clinical diagnostic studies. In this talk, I will discuss the parameters important in the design and performance of ddPCR for the detection and quantification of methylated DNA. I will present an example that demonstrates the sensitivity and precision of the method for detecting methylated DNA in the promoter region of mir342/EVL, a potential DNA methylation biomarker for colon cancer risk.

9:00 Novel Method to Detect MicroRNAs Using Chip-Based QuantStudio 3D Digital PCR

Orazio_FortunatoOrazio Fortunato, Ph.D., Experimental Oncology, Fondazione IRCCS Istituto Nazionale Tumori

Research efforts for the management of lung cancer are directed to identify new strategies for early detection. miRNAs are a new class of circulating biomarkers but lack of consensus on data normalization has affected their diagnostic potential. There is a growing interest in techniques that allow miRNAs absolute quantification. We described an interesting approach for profiling miRNAs in plasma samples using a chip-based QuantStudio 3D digital PCR. Given its reproducibility it would be a robust tool for quantitative assessment of miRNA for diagnosis of lung cancer and other diseases.

9:30 Mutant DNA quantification by digital PCR can be confounded by heating during DNA fragmentation

Qing Kang, Ph.D., Research Fellow, Internal Medicine, University of Michigan

Digital PCR (dPCR) is gaining popularity as a DNA mutation quantification method for clinical specimens. Fragmentation prior to dPCR is required for non-fragmented genomic DNA samples; however, the effect of fragmentation on DNA analysis has not been well-studied. Here we evaluated three fragmentation methods for their effects on dPCR point mutation assay performance.


10:00 Generalized Linear (and Mixed) Models for the Analysis and Design of Digital PCR Experiments

Bob_DorazioRobert M. Dorazio, Research Statistician, Biological Resources Division, United States Geological Survey

Generalized linear models of binomial responses and extensions of these models for extra-binomial sources of variation are appropriate for the analysis and design of experiments that use digital PCR (dPCR) to quantify nucleic acid concentration. These models are versatile and easy to fit using conventional statistical software. As illustrations, I analyze dPCR data from different kinds of experiments, including serial dilution, evaluation of copy number variation, and quantification of gene expression.

10:30 Coffee Break


11:00 Strategies and Challenges for Accurate Quantification of NGS Libraries with Digital PCR

Peter_SchweitzerPeter A. Schweitzer, Ph.D., Director, Genomics Facility, Institute of Biotechnology, Cornell University

The Cornell University Genomics Facility uses dPCR as an integral part of our Illumina sequencing operation for pooling samples and accurate quantification of final sequencing libraries. This has improved our ability to provide high quality DNA sequence data in a timely and cost efficient manner and avoiding costly reruns. One significant challenge is accurately performing the large dilutions required and I’ll describe an internal reference to correct for variability during dilution.

11:30 Using Picodroplet Digital PCR Technology on NGS Libraries to Create Rapid, Highly Sensitive Assays for Mutation Detection

Maria_ArcilaMaria Arcila, Ph.D., Director, Diagnostic Molecular Pathology Laboratory, Associate Attending Pathologist, Memorial Sloan Kettering Cancer Center

Although next generation sequencing (NGS) is a highly attractive technology for comprehensive assessment tumor samples, this approach alone may not provide sufficiently rapid and sensitive detection for some mutations in clinical practice. Dedicated single gene testing may lead to tissue exhaustion in some settings, precluding further assessment of the sample by NGS. We describe our clinical application of picodroplet digital PCR for rapid mutation detection using small aliquots of NGS libraries prepared for comprehensive profiling, fully maximizing broad genomic analysis on limited samples.


12:00 pm Utility and Advantages of Digital PCR Technique in Management of Hematological Malignancies

Rashmi_Kanagal-ShamannaRashmi Kanagal-Shamanna, M.D., Assistant Professor, Hematopathology, Molecular Pathologist, MD Anderson Cancer Center

The talk discusses the 1) principles of digital PCR 2) and potential applications in management of hematological neoplasms. The talk is specifically geared towards its applications in assessment of minimal residual disease in patients diagnosed with acute myeloid leukemia, myelodysplastic syndromes and myeloproliferative neoplasms.

Biorad12:30 Luncheon Presentation: Complementarity of Digital PCR and NGS for Liquid Biopsy

George_Karlin-NeumannGeorge Karlin-Neumann, Ph.D., Director, Scientific Affairs, The Digital Biology Center, Bio-Rad Laboratories

The choice of technology for a particular liquid biopsy application depends upon multiple factors including what information is already known about the patient’s disease and what needs to be learned for effective care. Where a wide range of genetic or epigenetic markers may be driving initial or recurrent disease, the broad profiling approach offered by NGS is beneficial. However, when a limited set of gene variants are driving tumor growth or tagging tumor or organ turnover—and particularly when serial monitoring of known markers is desired --digital PCR offers a more rapid, sensitive, specific and economic approach. Examples of this complementarity will be presented from both the oncology and transplant monitoring fields.

1:00 Session Break


2:00 Chairperson’s Remarks

Maria Arcila, Ph.D., Director, Diagnostic Molecular Pathology Laboratory, Associate Attending Pathologist, Memorial Sloan Kettering Cancer Center

2:05 Early Response Monitoring with Circulating Tumor DNA in Lung Cancer

Hatim_HusainHatim Husain, M.D., Assistant Professor, Medicine, Hematology and Oncology, University of California, San Diego

Non-invasive drug response biomarkers for early assessment of tumor response can enable adaptive therapeutic decision-making for individualized patient treatment and proof-of-concept for target inhibition of tumor cells by investigational drugs. Circulating tumor DNA (ctDNA) is released into the blood by tumor cell turnover and excreted into urine. Sampling flexibility of urine was leveraged to determine whether detection and quantitation of ctDNA biomarkers in urine could assess early tumor response within days of a patient receiving targeted therapy. Findings suggest that tyrorsine kinase inhibitors induced tumor apoptosis within days of initial patient dosing. We demonstrate that daily monitoring of ctDNA by non-invasive urine sampling provides temporal and quantitative dissection of early tumor response.

2:35 Liquid Biopsies in Biomarker Driven Cancer Care

Filip_JankuFilip Janku, M.D., Ph.D., Assistant Professor, Investigational Cancer Therapeutics (Phase I Program), MD Anderson Cancer Center

The optimal choice of cancer therapy depends upon analysis of the tumor genome for molecular alterations. The tumor heterogeneity of cancers creates substantial challenges, as molecular profile depends on time and site of tumor tissue acquisition. To capture the entire molecular profile, multiple biopsies from primary and metastatic sites at different time points would be required, which is ethically and economically problematic. Molecular analysis of circulating cell-free DNA offers a novel, minimally invasive method that can be performed at multiple time-points and plausibly better represents the prevailing molecular profile of the cancer. Molecular analysis of this cell-free DNA offers multiple clinically useful applications, such as identification of molecular targets for cancer therapy, monitoring of tumor molecular profile in real time, detection of emerging molecular aberrations associated with resistance to particular therapy, determination of cancer prognosis and diagnosis of cancer recurrence or progression.

3:05 Refreshment Break

3:35 Digital PCR for Infectious Disease

Alexandra_WhaleAlexandra Whale, Ph.D., Senior Researcher, Molecular and Cell Biology Team, Science and Innovation Division, LGC

It has been suggested that digital PCR may offer a number of advanced applications that potentially enable improved detection and management of infectious diseases. To test this, we evaluated its performance in the detection of a range of microbial targets, including oseltamivir resistance in Influenza.

4:05 FEATURED PRESENTATION: Droplet Digital PCR for Microbial Risk Assessment in Beach Water

John_GriffithJohn Griffith, Ph.D., Principal Scientist, Southern California Coastal Water Research Project

Molecular methods have become routine in clinical applications, but are recent additions to the environmental monitoring and risk assessment toolbox. This talk will describe how digital PCR is being utilized to monitor water microbial water quality, identify sources of fecal contamination and quantify pathogens for risk assessment, and how a flow-through digital PCR instrument now under development has the potential to revolutionize the field of water quality testing.

4:35 Close of Symposium

Click here to see who's attending the symposium.