by George Karlin-Neumann


Life Science


Enabling Cancer Research and Treatment With Droplet-Based Digital PCR Technology


C


ancer research is as necessary as ever, especially if we are to fulfill the promise of personalized (or “precision”) medicine based on specific cancer therapies directed to known molecular targets. Fortunately, discovery is possible at a faster and more efficient pace with the help of innovative new research techniques.


Every year, roughly 14 million people learn they have cancer, and eight million people die from the disease, according to global statistics from the Centers for Disease Control and Prevention. However, research sug- gests that one-third of cancer deaths can be prevented with the right tools and education.


Droplet digital PCR One emerging tool is a pioneering technology called droplet digital


PCR™ (ddPCR™) (Bio-Rad Laboratories, Pleasanton, CA). In the past year and a half, a growing number of cancer researchers have begun using ddPCR for its ability to interrogate the cancer genome with un- precedented levels of sensitivity and precision. Although it is not the first implementation of digital PCR (dPCR), its unparalleled performance, quick turnaround time, and affordability are causing scientists to increas- ingly recognize its potential as a tool in the detection and monitoring of cancer, which can ultimately save lives.


The power of partitioning—precision,


sensitivity, and robustness First, let’s look closer at what dPCR entails and why it is advantageous for researchers today. At its core, dPCR is a method of counting nucleic acid molecules. It works by partitioning the sample in such a way that either zero or only a small number of nucleic acid molecules of interest (i.e., targets) are present in each partition.


This partitioning happens by dividing the sample into compartments (or partitions)—either microfluidic chambers or droplets, though his- torically, PCR-plate wells were used. The sample is then amplified to endpoint using normal PCR conditions (in a conventional thermocycler for ddPCR). An absolute measurement of the target concentration is achieved by scoring the positively and negatively fluorescent partitions. Unlike quantitative real-time PCR (qPCR), dPCR eliminates the need for a standard curve.


Eliminating the standard curve reduces error and improves precision in ddPCR. This enhanced precision enables day-to-day and lab-to-lab reproducibility, a longstanding weakness of prior methods. In addition,


because samples are partitioned, the impact of competing background DNA is reduced, allowing for greater discrimination between similar sequences. In addition, because reactions are run to endpoint, they are more tolerant of potential sample inhibitors.


Introduced in 2011, the QX200 Droplet Digital PCR (ddPCR) system from Bio-Rad has become the most widely adopted approach to dPCR—with peer-reviewed publications describing use of the technology now appearing at a rate of about one every three days. It offers greater ease- of-use, enhanced performance, higher throughput, and lower costs compared to previous dPCR implementations, such as fluidic chip-based methods. The ddPCR system partitions samples into 20,000 droplets per well (in a 96-well plate), enough to give sufficient single-well precision and sensitivity for most high-throughput applications (300 samples/ day), but with the flexibility to merge sample wells in silico when still greater precision or sensitivity is desired.


Empowering cancer research dPCR is being used in basic research, translational research, and clinical


cancer research. In basic research, ddPCR allows researchers to examine tumor heterogeneity, clonal evolution, and metastasis, and to answer cell origin questions. In translational and clinical settings, ddPCR is being used to monitor drug resistance, identify alternative druggable targets, and prevent overtreatment, as well as measure dose-responses in tar- geted therapy underlying personalized medicine treatments.


Rare cancer mutation detection is one of the most common applications for ddPCR—used to test prognostic and predictive indicators, monitor residual disease, and preselect patients for clinical trials. Copy number variation (CNV) identification is another common ddPCR application. CNVs are structurally variant regions that involve either gains or losses of genomic DNA that have been shown to be associated with cancer.


The level of sensitivity offered by the Bio-Rad ddPCR system is also help- ing researchers quantify cancer biomarkers. Thus, ddPCR could be the key to bringing liquid biopsies—one of medicine’s holy grails—closer to clinical reality. These tests use blood or other body fluids such as urine instead of tissue from traditional biopsies to detect cancer, track its progress, and guide treatment decisions. ddPCR is able to isolate one molecule of mutant DNA into a droplet with just a few corresponding wild-type molecules, thus making the mutant DNA more detectable. What was previously undetectable with other methods can now be quantified.


AMERICAN LABORATORY • 27 • SEPTEMBER 2014


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