Positive Droplet Award Program

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Allen KC Chan, PhD

Professor of Chemical Pathology
Chinese University of Hong Kong

About Professor Chan

Professor Allen Chan is currently Chair Professor of Chemical Pathology. His research focus is on the development of novel diagnostic approaches using circulating DNA. With Professor Dennis Lo, he developed non-invasive prenatal tests (NIPT) for Down syndrome and monogenic conditions, as well as early detection of multiple cancers. In 2009, he also pioneered the use of digital PCR for the detection of EGFR mutations. These approaches have been adopted for clinical use. Professor Chan is a co-inventor of over 150 patent families and is listed as Top 20 Translational Researcher by Nature Biotechnology for 2020.

Professor Chan’s Key Publications

• Single-molecule Detection of Epidermal Growth Factor Receptor Mutations in Plasma by Microfluidics Digital PCR in Non-small Cell Lung Cancer Patients
• Liver- and Colon-specific DNA Methylation Markers in Plasma for Investigation of Colorectal Cancers with or without Liver
• DNA of Erythroid Origin is Present in Human Plasma and Informs the Types of Anemia

Impact of ddPCR on Professor Chan’s Research

My research focus is to develop new applications for circulating cell-free DNA analysis covering areas of noninvasive prenatal testing (NIPT), oncological applications, and transplant monitoring. In these applications, the DNA targets (i.e., the DNA derived from the fetus, the tumor, and the transplanted organ) exist at very low concentration in blood plasma, together with a background of DNA derived from blood cells. To sensitively detect and accurately quantify these targets is a challenging task. The invention of digital PCR has led to significant improvement in both the sensitivity of detection and precision in the measurement of DNA targets present at low concentrations. In 2009, we demonstrated that the detection of Epidermal Growth Factor Receptor (EGFR) mutations in the plasma of lung cancer patients can accurately reflect the EGFR mutational status in the corresponding tumor tissues and hence predicts the response to EGFR tyrosine kinase inhibitor treatment. We also demonstrated that digital counting of plasma DNA molecules could provide unprecedented precision for detecting chromosomal aneuploidies. Based on this principle, we eventually developed the widely used noninvasive prenatal test for Down syndrome. In our latest work, we demonstrate that droplet digital PCR can be combined with next generation sequencing to enable new applications in NIPT and pharmacogenomic analysis.

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Jim Huggett, PhD

National Measurement Laboratory
University of Surrey

About Professor Huggett

Dr. Huggett is a Science Fellow at the National Measurement Laboratory (NML, the UK's designated national measurement institute for chemical and bioanalytical measurement based at LGC). Much of his interests are focused on accuracy for molecular analysis, reference measurement systems for nucleic acid measurement and improving the accuracy of applied routine diagnosis and he has published widely on this topic and also led the writing of the digital MIQE guidelines.

Professor Huggett’s Key Publications

• Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine
• An assessment of the reproducibility of reverse transcription digital PCR quantification of HIV-1
• The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020

Impact of ddPCR on Professor Huggett’s Research

I have been working with digital PCR for over a decade and see some real benefits offered by this technology. Commonly described advantages include increasingly sensitive rare variant, or high precision detection that allow for measurements that are either not possible or more complex using alternative methodologies. However, for me one of the main, yet unsung, advantages of digital PCR is its high reproducibility when measuring absolute numbers of nucleic acid molecules. It is now simply much easier to get reproducible results across multiple labs when using this technology. This could have a huge impact on the translation of research findings to applied scenarios where high reproducibility is essential while also supporting the transition of molecular biology into an increasingly precise science.

Digital PCR also has the potential of measuring nucleic acids with unprecedented accuracy and part of my work has been investigating how to achieve this. This is made possible as digital PCR is can, under certain situations, detect all the amplifiable molecules present in a reaction and by highly accurate estimation of partition volumes. We are now confident that, in certain situations, we can make International System of Units (SI) traceable measurements using digital PCR to quantify nucleic acid extracts. We are now investigating how to best apply this capability both directly, but also in support of other methods as a reference measurement procedure to value assign reference materials and define reference ranges. While this subtle advantage may not at first appear that revolutionary, or not as exciting as some of the other potential applications, I believe it could be the biggest game changer as we have never really been able to make absolute measurements with this level of accuracy before; similar breakthroughs in other areas of analytical science have often opened doors to new possibilities.

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Deendayal Patel, PhD

Principal Scientist, Molecular Analytics
Bristol Myers Squibb

About Dr. Patel

Dr. Patel is a Principal Scientist at Bristol Myers Squibb engaged in molecular analytical development for CAR T-cell drug products. He has developed a ddPCR-based assay that is capable of detecting multiple attributes and can be applied at any stage of the CAR T-cell process. Dr. Patel received his PhD in virology in India and did his post-doctoral training in molecular virology at UMD-College Park, MD. He has also done research at SUNY-Buffalo, NY and Rutgers, NJ.

Dr. Patel’s Key Publications

• Inhibition of primary effusion lymphoma engraftment in SCID mice by morpholino oligomers against early lytic genes of Kaposi’s sarcoma-associate herpesvirus
• Porcine reproductive and respiratory syndrome virus inhibits type I interferon signaling by blocking STAT1/STAT2 nuclear translocation
• Morpholino oligomer-mediated protection of porcine pulmonary alveolar macrophages from arterivirus-induced cell death

Impact of ddPCR on Dr. Patel’s Research

In CAR T-cell therapy, accurate and rapid measurement of vector copy number (VCN) and replication competent lentivirus (RCL) are important quality control steps required for the release of CAR T-cell products for patient infusion. My group was using a qPCR-based standalone assay that was already established within our molecular analytics unit for detection of both the targets (i.e., VCN and RCL). However, the technological advancement in the CAR T-cell therapy, such as bispecific CAR or allogenic CARs, imposed challenges in analytical development and it became important to modify the already established analytical methods so that multiple additional attributes could be evaluated within the same sample. Flow cytometry-based assays can be used for monospecific CARs identification. However, in case of bispecific CAR that has two different drug targets, the identity assessment by flow cytometry becomes very challenging therefore we needed a molecular method. Additionally, the manufacturing of both established monospecific CARs and bispecific CAR, which is derived from monospecific CAR, is carried out in the same facility and thus, it was important to have an identification method that is differential for each drug product (DP). Another challenge was to differentiate and detect the bispecific CAR-T cell DP in patient who has history of prior CAR T-cell therapy. The lentivirus and retrovirus vectors used in the CAR T-cell therapy have the capacity to integrate permanently into host cell DNA increasing the risk of oncogenesis if the vector copy number (VCN) per cell is high. Regulatory agencies recommend that the VCN should be <5 copies per genome so we needed a reliable method that can precisely detect the low VCN in the patient samples.

We started to explore different avenues to advance our analytical development technology which can align well and fulfil the business needs. In ddPCR, I most like the absolute quantification and the multiplexing functions. Initially I developed a single-plex VCN assay on QX200 instrument. The assay worked well, and the results were very specific, with high precision and sensitivity and that gave us the confidence to pursue this further. Moving forward, we switched to the QX ONE ddPCR system which is fully automated, and it can simultaneously detect 4 channels. I would say QX ONE is more biotech or pharmaceutical industry friendly so far. I was able to develop a multiplex ddPCR assay that can detect simultaneously 4 targets in single assay-VCN, CAR T-cell identification, VCN from existing therapy, and RCL. This method has been successfully validated for bispecific CAR T-cell DP. In the absence of the multiplex ddPCR assay that I developed we would have to perform multiple assays to verify each target. Thus, the test developed has improved the turnaround time and is cost-effective. Moreover, there is ample opportunity to refine this multiplex ddPCR-based approach according to target necessity. This advancement of the analytical methods will ultimately support the goal of our CAR T-cell therapy, which is to deliver more medicines to more patients, faster.