NEW YORK (GenomeWeb) – In the past few years, there has been a steady increase in the number of published peer-reviewed studies using commercial droplet-based digital PCR platforms, suggesting acceptance and uptake of the technology for molecular biology research.
Due to its unique capabilities, particularly its ability to detect rare events, the technology is also paving the way for cutting edge clinical applications such as liquid biopsies, recent publications suggest.
In the digital PCR space, two companies make droplet-based digital PCR (ddPCR) systems in which PCR is performed in microdroplets separated by oil — Bio-Rad manufactures the QX100 and QX200 Droplet Digital PCR systems, while RainDance Technologies makes the RainDrop ddPCR platform.
Meanwhile, two other companies manufacture systems that use a plate format — Thermo Fisher brand Life Technologies makes the OpenArray and QuantStudio platforms, while Fluidigm makes the Biomark HD featuring integrated fluidic circuit technology.
Droplet-based systems are younger than plate-based systems, with the Bio-Rad and RainDance systems launching within the past four years. And, due to the sheer number of reaction volumes they can generate, droplet-based systems are capable of some things that plate-based systems are not. Early adopters of ddPCR are now publishing some of these applications at an increasing rate.
In 2013, a total of 49 peer-reviewed studies used Bio-Rad's ddPCR platforms, and in 2014 that number more than doubled to 118 studies, according to a Bio-Rad spokesperson. Meanwhile, the RainDance system has been on the market for less time, and the company highlights eight ddPCR publications on its website, although CEO Roopome Banerjee told GenomeWeb that more than 50 publications have featured RainDance's droplet technology. RainDance also uses its droplet technology in a dedicated platform for target enrichment in next-generation sequencing.
"Peer-reviewed ddPCR articles are [now] emerging at a rate of a few per week," George Karlin-Neumann, director of scientific affairs at Bio-Rad's Digital Biology Center, said in a recent email to GenomeWeb.
"The creativity and vetting of Droplet Digital PCR by early adopters is convincing the larger scientific (and policy) communities that it is a valid and reliable tool for both basic and applied research," he said, adding that there are "knock-on effects" of having so many systems in use around the world with a high level of overall productivity.
"More and more people have access to the QX systems, and more and more of their colleagues are demonstrating the diverse areas of research that are enabled or enhanced by the technology," Karlin-Neumann said.
Bio-Rad obtained its ddPCR technology via a $162 million acquisition of QuantaLife in 2011. It began shipping QX100 Droplet Digital PCR systems in January of 2012. Sales of the platforms were slower than anticipated initially, but picked up through the first year.
The firm launched a next-generation version, the QX200, in 2013, and an automated droplet generator in late 2014.
Bio-Rad CEO Norman Schwartz announced at the JP Morgan Healthcare Conference in January of 2013 that the company had placed more than 200 systems worldwide by that point. But, in an earnings call the following month, Christine Tsingos, executive vice president and CFO, declined an analyst's request to disclose exact sales figures for the digital PCR business, noting the company's policy to not break out revenues for specific product lines. Bio-Rad's ddPCR business also remained on a growth trajectory through 2013.
RainDance, meanwhile, launched the RainDrop system in 2012 via an early-access program, and began placing platforms in labs in 2013. It has since obtained $35 million in financing to support growth of its ddPCR business, the company said.
Regarding RainDance's install base, Banerjee said the company has placed more than 100 systems thus far.
According to one review, although digital PCR in general liberates researchers from the standard curves of real-time PCR, thereby potentially increasing reproducibility within and between labs, plate- and chip-based versions of the technology are still limited in their usefulness in a clinical diagnostic setting. "Some of the commercial dPCR platforms have a relatively small number of partitions that can only be scaled up using multiple costly, one-time-use microfluidics chips," the authors wrote.
Droplet-based platforms, however, may hold more promise for clinical applications, which both Bio-Rad and RainDance seem to be targeting in recent months.
Bio-Rad has launched a line of kits using ddPCR for mutation and copy number variation detection, and debuted a multiplexed KRAS detection kit this week. The kit has reagents for 200 reactions, and a list price of $2,000, according to the company's website. Two other kits for CNVs are "coming soon," and the firm also sells hundreds of primer and probe sets for clinically relevant ddPCR under its PrimePCR brand.
However, according to RainDance CEO Roopom Banerjee, Bio-Rad has been primarily positioning its platform as an alternative to qPCR, while RainDance has elected to focus its attention one main application: liquid biopsy.
"We're in a more focal, exciting, and strategically valuable market, [while] they're going after a large, established … 20 year-old market," Banerjee said. "One would naturally expect that given those differences in strategic positioning and their head start, that they would have more publications," he said.
Basic research trends
Bio-Rad's Karlin-Neumann noted a number of trends among the recent ddPCR publications.
For example, studies detecting copy number variation and mosaicism seem to be on the rise. He highlighted one study of a CNV associated with hypertension, one describing parental transmission of microdeletions, and another showing mosaicism among human skin cells.
Another trend is the validation of next-generation sequencing results showing CNVs, RNA edits, or the combination of gene expression and RNA edits such as in the personal omics profiling of the "Snyderome." These NGS applications are paralleled by Bio-Rad's acquisition of sequencing technology firm GnuBio last year.
Other trends include use of EvaGreen-based methods for studying alternative splicing in cancer, as well as ontology tracing, cell counting, and high-sensitivity expression analyses, including such feats as measuring single-cell genetic variability underlying fetal brain cell response to environmental stress.
More groups also seem to be taking advantage of the technology's ability to overcome PCR inhibition.
In this vein, Karlin-Neumann pointed out a recent publication describing quantification of soil bacteria over time to analyze microbial ecology. Another showed ddPCR's usefulness in distinguishing general fecal indicators from human-associated fecal indicators for water quality assessment. That study noted that digital PCR "has the potential to become a reliable and economical alternative to qPCR for recreational water monitoring and fecal source identification," and that findings from the study "may also be of interest to other aspects of water research such as detection of pathogens and antibiotic resistance genes."
Also in this bin is a study of preclinical Alzheimer's disease that used ddPCR to perform absolute quantification of a mitochondrial DNA marker in cerebrospinal fluid, a sample type that can have PCR inhibitors such as endogenous polymerase inhibitors and heme products from breakdown of erythrocytes. In this case, ddPCR also circumvented error-prone qPCR standard curves, Karlin-Neumann noted.
Overcoming PCR inhibitors is also an important factor in detecting genetically modified organisms, as demonstrated in a study of food and feed samples, as well as in food processing for pathogens. Bio-Rad's QX100 was adopted by GMO testing company Genetic ID in 2013, as previously reported.
Another major trend is the increasing use of ddPCR for rare event detection, including attempts to measure rare cancer biomarkers or viral eradication.
GenomeWeb has covered this uptake extensively, reporting on studies using ddPCR on circulating microRNA to detect lung cancer, circulating tumor cells to detect cervical cancer, and studies measuring T-cell response in tumors, and BCR-ABL1 fusion transcripts. The platform is now also being used by startup Toma Biosciences to detect cancer-related mutations in formalin-fixed, paraffin-embedded tissue.
Karlin-Neuman said that the Bio-Rad platform was used in a recent study to detect cancer DNA in plasma from patients with early stage breast cancer, for real-time monitoring and treatment optimization of a patient with melanoma and occult leukemia, and for BCR-ABL detection in children with chronic myeloid leukemia.
Publications describing latent, or low viral load HIV detection using ddPCR were already on the rise a year and a half ago, as GenomeWeb reported in a previous review of the technology.
Although initial reports suggested that ddPCR did not show significant advantages over qPCR for HIV detection, and that false positive droplets could interfere with low level detection in viral eradication studies, two recent studies have recently described improved sensitivity.
Karlin-Neuman also noted an HIV viral eradication study that used the QX100 to measure gene editing in CD4 T-cells.
Meanwhile, other researchers are attempting to use ddPCR to determine hepatitis B eradication, a method they also mathematically modeled. That group, however, has also determined that, in terms of viral diagnostics, adopting ddPCR for cytomegalovirus monitoring may still be premature.
Karlin-Neumann also pointed to a number of non-cancer blood screening studies, including pre-diabetes screening, previously covered by GenomeWeb, and early diagnosis of sepsis.
On the so-called liquid biopsy front, meanwhile, a clinical trial is underway at the Dana-Farber Cancer Institute using ddPCR on plasma samples for non-invasive tumor genotyping for melanoma and lung cancer. This group has also published on non-invasive detection of treatment response and resistance in EGFR-mutant lung cancer, while another group used ddPCR on plasma samples to non-invasively measure BRAF-inhibitor response in melanoma.
This liquid biopsy market is also RainDance's primary focus.
Here, Banerjee suggested the RainDrop platform — which can multiplex up to 10 channels and runs 10 million droplets versus Bio-Rad's 20,000 — may have an advantage in rare event detection and absolute quantification. "We are the only true single-molecule digital PCR player out there and we don’t tolerate multiple occupancy in droplets," he said.
Liquid biopsy is carving out a space between translational and pharma, Banerjee said. "As a company we've seen very broad demand across major cancer centers, the pharma companies, as well as the medical genetic centers in the US," he said.
RainDance's platform has a number of early adopters, and was used in a few recent studies, including one detecting KRAS mutations in plasma from colorectal cancer patient, and another on CRC biopsies from patients with KRAS mutations who were treated with anti-EGFR therapy.
Banerjee also noted that between 30 and 40 percent of the RainDance's customers are adopting both its platform and the Bio-Rad platform side-by-side. Users might do this because they've "hit the sensitivity ceiling" with Bio-Rad's ddPCR, they have a need for both low- and high-sensitivity platforms, or possibly because of Bio-Rad's positioning as a qPCR alternative versus RainDance's liquid biopsy emphasis, Banerjee said.
Most of the clinical applications for ddPCR are being explored in the context of laboratory-developed tests. An obvious next step for this technology would be for a platform to get approval from the US Food and Drug Administration to use for molecular diagnosis.
"It's not only possible, but in fact highly probable that most of us will go down that path at some point in time," Banerjee said.
"I think as an industry we're starting to wrap our heads around the size and scale of the opportunity, and everyone is developing the validation clinical database to be able to support the value of these technologies in clinical use long term," he said, noting that the a core advantage for ddPCR firms is FDA familiarity with PCR.
"The FDA has cleared over 500 PCR tests in its history, and so [it has] a high level of knowledge and familiarity with PCR-based tests ... getting a test through the FDA ultimately should follow very well-known, established paths."