Patricia F. Fitzpatrick Dimond Ph.D. Technical Editor of Clinical OMICs President of BioInsight Communications

Could dPCR be a diagnostics dark horse?

Over the past few years, scientists from a variety of disciplines have applied digital PCR (dPCR) as a potentially useful tool for breast cancer screening, measuring latent HIV reservoirs in patients, and diagnosing hospital-acquired and sexually transmitted infections, among others. Digital PCR and variations on it offer a new approach to nucleic acid detection and quantification.

Similarly to real-time quantitative PCR (qRT-PCR or qPCR), dPCR carries out a single reaction within a single sample. However, in dPCR the sample is separated into a large number of partitions, and the reaction is carried out in each partition individually. This separation, proponents of the technology say, allows a more reliable collection and sensitive quantitation of nucleic acids.

And a recent advance in dPCR, droplet digital PCR (ddPCR™), can measure absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions. The technology is said to offer advantages in the field of liquid biopsies, enabling circulating nucleic acids (cfDNA) and circulating tumor cells (CTCs) to be measured in blood. The technique can also detect rare tumorigenic mutations in a high background of “normal” DNA, routinely down to 0.01% and often further.

But although simple in theory and principle, the technique’s implementation was not, as it was carried out in commercially available 384-well plates with five microliters per partition, requiring large volumes of reagents.

The dPCR Arena

Advanced nanofabrication and microfluidics technologies have now been incorporated into systems that produce hundreds to millions of nanoliter- or even picoliter-scale partitions, but to date, few companies have jumped into the dPCR arena, offering instrumentation platforms that perform dPCR, or both qPCR and dPCR in various configurations.

In 2006, Fluidigm became the first company to commercialize dPCR. Currently it offers two systems that mix samples with reagents, partition the reaction mixture, and perform thermocycling and read results within each partition. The systems use chips containing microfluidics and valves that partition samples into about 800 reactions, with either 12 or 48 samples per chip.

Life Technologies, through its 2009 acquisition of BioTrove, now offers two machines that can be used for both digital PCR and qPCR, the OpenArray and QuantStudio 12K Flex. These mix samples with reagents, load mixtures into reaction chambers, run amplification cycles and monitor reactions as they occur. The machines rely on plates that are roughly the size of microscope slide boards with nano-sized holes; capillary forces and careful placement of hydrophilic and hydrophobic surfaces hold samples in place.

Life Technologies has also introduced the QuantStudio 3D Digital PCR System. This system, which began shipping in June 2013, is built around a high-density, nanofluidic silicon chip that enables up to 20,000 data points. The system’s chip-based approach is meant to simplify workflow, decreasing the number of hands-on steps needed to begin experiments, and reducing the risks of sample contamination and loss of DNA.

Companies like Bio-Rad and RainDance now market instruments with many more partitions than previously possible using plates with nano-sized holes. In droplet digital PCR, reaction chambers are separated not by the walls of a well but by carefully titrated emulsions of oil, water, and stabilizing chemicals. Samples are put into a machine where they are mixed with all the necessary reagents and dispersed into tiny droplets. The droplets for each sample are transferred into tubes that can be placed in a thermocycler for PCR. Afterward, the tubes are transferred to a droplet-reading machine, which functions like a flow cytometer to analyze each droplet for whether or not a reaction has occurred.

RainDance, for example, has developed a patented way to put reagents inside of picoliter-sized droplets to encapsulate biology one droplet at a time. Currently, single nucleic acids are placed inside of the droplets. This creates a single-plex PCR reaction inside of each droplet and, the company says, droplets can be generated using one of RainDance’s commercial instrument systems at up to 10,000 per second.

In an email to GEN, George Karlin-Neumann, Ph.D., director of scientific affairs at the Digital Biology Center, Bio-Rad clarified distinctions between qPCR and his company’s QX200 Droplet Digital PCR™, explaining that either system can quantitate DNA or RNA targets with either Taqman 5’ nuclease assays or fluorescent DNA-binding dyes (SYBR for qPCR, and EvaGreen for ddPCR) run in suitable Master Mixes.

But, he explained, ddPCR divides a sample reaction into many thousands of small, uniformly sized droplets where each may or may not contain a target template of interest. After thermocycling to endpoint in a standard 96-well plate and thermocycler, the droplets in each well are read and counted in a droplet flow cytometer (or reader) to determine which droplets have the target (“positive” droplets) and which do not (“negative” droplets). The fraction of positive droplets reflects the number of target molecules in the reaction volume, thus yielding the concentration measurement sought.

dPCR and Cancer Detection

And researchers have adopted dPCR for numerous applications including for analysis of several parameters in cancer patients. In study results published in Clinical Cancer Research last March, researchers from the Royal Marsden Hospital in London described their adaptation of ddPCR to determine the presence of oncogenic amplification through noninvasive analysis of circulating free plasma DNA and exemplify this approach by developing a plasma DNA digital PCR assay for HER2 copy number.

Because HER2 copy number in digital PCR is assessed relative to a reference gene, the investigators used EFTUD2, a gene within the ERBB2 locus found not to co-amplify with HER2 and not subject to normal copy-number variations.

Using the Bio-RAD QX100 ddPCR system, the researchers found that 64% of patients with HER2-amplified cancers were classified as digital PCR HER2-positive and 94% of patients with HER2-nonamplified cancers were classified as HER2-negative by the assay, giving a positive and negative predictive value of 70% and 92%, respectively.

The authors concluded that “digital PCR of plasma DNA has high accuracy in the determination of HER2 status,” and that the approach of analyzing of plasma DNA with digital PCR has the potential to screen for the acquisition of HER2 amplification in metastatic breast cancer. “This approach could potentially be adapted to the analysis of any locus amplified in cancer,” they concluded.

And last September, scientists working at Fred Hutchinson Cancer Research Center, demonstrated that ddPCR technology could be used to precisely and reproducibly quantify microRNA (miRNA) in plasma and serum over the course of different days, potentially allowing further development of miRNA and other nucleic acids as circulating biomarkers.

Under active study as blood-based biomarkers for cancer and other diseases, miRNA measurements in blood samples have been plagued by unacceptably high interday variability, obviating their use as reliable blood-based biomarkers.

“In the field of circulating microRNA diagnostics, droplet digital PCR enables us to finally perform biomarker studies in which the measurements are directly comparable across days within a laboratory and even among different laboratories,” said Muneesh Tewari, M.D., Ph.D., associate member in the Human Biology Division at the Fred Hutchinson Cancer Research Center and lead author of the study.

And Dr. Karlin-Neumann says that ddPCR is “en route to being introduced into clinical practice in a number of areas.” Though, he notes, the “only CLIA lab I know of that currently offers a ddPCR-based test is the University of Washington’s Clinical Laboratory, which offers a ddPCR-based test for detection of chromosomally integrated HHV-6 virus in transplant patients.”

Other labs, he says, that are in the process of developing clinical tests for detection of residual disease in leukemia patients with BCR-ABL translocations include that of Alec Morley, M.D., a pioneer of digital PCR. Dr. Karlin-Neumann also cites the work of Hanlee Ji, M.D., who is measuring copy-number variations by ddPCR in FFPE and cell-free plasma DNA to assess whether gastric and other cancer patients have amplifications in oncogenes that would make them amenable to one of a growing number of targeted therapies.

Importantly, Dr. Karlin-Neumann pointed out that it’s still too early, regardless of the platform used, to be attempting to detect cancer in naïve patients not already known to have cancer since “we do not have the clinical experience to know what changes to look for and what thresholds are meaningful.”

And he notes, until recently, there have not been technologies that allowed us to detect and quantitate below ~1% mutant abundance in either mixed tissue biopsies or in cfDNA in plasma or serum. ddPCR is demonstrating that it is capable of lowering this limit to as low a ~0.01% in a single ddPCR reaction well, and where more material is available, this can be lowered further by use of multiple wells. Similarly, fractional changes in oncogenic amplifications and deletions can be tested with ddPCR in both solid tumors and in cfDNA.

And a team of scientists at the University of California, Berkeley says it has developed a bead-based, microfluidic digital PCR technology and demonstrated its ability to quantitatively measure cancer-related translocation mutations at extremely low levels and subsequently sequence single mutated clones.

The scientists believe that their technology has advantages over commercial emulsion-based droplet digital PCR platforms, such as those offered by Bio-Rad and RainDance Technologies, because it enables downstream sequencing analysis following the digital PCR analysis step.

But is this capability in demand? A RainDance spokesperson told PCR Insider that the company’s RainDrop digital PCR system currently does allow for emulsions to be broken following thermal cycling so the amplicons can be rescued and subsequently sequenced. However, RainDance said, it is “just starting to see requests for this kind of thing but it is not a commercial solution on offer at this point.”

But technology will get continue to get piled higher and deeper, as modifications to PCR continue to accrue and scientists figure out how best to use them.

Patricia Fitzpatrick Dimond, Ph.D. ([email protected]), is technical editor at Genetic Engineering & Biotechnology News.

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