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As RainDance Readies to Place Digital PCR Systems, Early Users Weigh in on Platform's Potential

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It has been nearly a year since RainDance Technologies officially "launched" its digital PCR platform, RainDrop, by recruiting researchers to be part of an early-access program.

And although it has taken more time than the company anticipated to begin placing the instruments in laboratories, RainDance finally expects to begin doing so this coming March, having garnered about a dozen purchase orders from customers and generated nearly 100 "active sales engagements," Andy Watson, chief marketing officer and head of global sales for the company, told PCR Insider recently.

Despite the delay, which Watson chalked up primarily to fine-tuning of the platform's closed-tube design, the platform has engendered significant interest from both the research and clinical diagnostics community.

Several early-access users and potential customers recently weighed in on the perceived benefits of the platform and how they plan to implement it, providing a clue about the most promising markets for the system and for droplet-based digital PCR in general.

RainDance first hinted in mid-2010 at the promise of its RainStorm picodroplet technology — which had from the company's start served as the basis of its RDT 1000 and later ThunderStorm targeted sequencing systems — for digital PCR applications (PCR Insider, 7/8/2010).

Last March the company officially unveiled such a platform, the RainDrop, at the American Association for Cancer Research meeting in Chicago (PCR Insider, 3/29/2012). By that point it had become clear that, although the digital PCR market had rounded into shape with offerings from vendors such as Fluidigm and Life Technologies, the RainDrop would most closely compete application-wise with the Bio-Rad QX100 Droplet Digital PCR system, acquired with QuantaLife, because of the similar droplet-generation technology employed by those systems.

But while Bio-Rad has begun to see healthy uptake of its digital PCR system (see related story, this issue, and PCR Insider, 12/20/2012); and reported in November that it more than doubled digital PCR sales in the third quarter over the second quarter to $5.2 million in receipts (PCR Insider, 11/8/2012), RainDance had yet to begin generating revenues from its platform.

"People have either been coming in [to RainDance's headquarters] to use it here … or sending samples to us to run assays," Watson said. "And we've had lots of internal projects with a dedicated team of in-house scientists."

The platform mostly needed refinement in the area of its closed-tube architecture. "One of the features that we saw was important to the marketplace was this idea that you have a closed tube from sample to result, much like qPCR does — you don't have to open your tube and pipette the solution out in order to read it," Watson said. "We implemented that design, and in testing that we found a few bugs that we had to fix … but it's now working well."

In the meantime, a number of potential RainDance customers — both early-access users and those who have been sending samples to the company — have had a chance to explore several features of the platform that RainDance believes provide an advantage over the QX100. Among the advantages claimed by the company are cost; smaller, more uniform, and more numerous individual droplet reaction volumes; multiplexing ability; and open chemistry.

"In terms of competition, we don't hear much in the way of Fluidigm or Life Tech," Watson said. "Customers coming to use either currently have a Bio-Rad system and want a replacement or a supplement, or are just beginning to look at [droplet-based digital PCR] and like what they see in [RainDance]."

One such customer is Michael Griffiths, professor at the School of Cancer Sciences at the University of Birmingham and director of the West Midlands Regional Genetics Laboratory at Birmingham Women's Hospital — the largest National Health Service diagnostic laboratory in the UK, covering about a tenth of the UK's population.

According to Griffiths, his lab has been in discussions with RainDance regarding both its targeted sequencing platforms and digital PCR system. Griffiths referred to RainDance's RDT 1000 platform, which his lab has been using on loan from RainDance, as "digital PCR;" and to the RainDrop platform as "droplet digital" PCR.

Griffiths said that since his lab is heavily involved with next-generation sequencing for various clinical applications and development projects, it has been assessing a variety of technologies, including the RDT 1000, for NGS library preparation.

"If we've got lots of patients where we want to look at a few genes per patient, then actually, we're …. typically using a Fluidigm Access Array 48.48 … [to look at] 48 amplicons for 48 patients at the same time," Griffiths said. "But if you've got a few patients with lots of targets, that's where the RainDance digital PCR interested us, because, [for instance], it allows us to design a myeloid leukemia panel of 56 genes with over 1,200 amplicons. That's what we're currently doing this evaluation work on at the moment."

Griffiths said that if the results it is achieving on the RDT 1000 continue to hold up, the group will consider switching to the higher throughput ThunderStorm platform.

On the droplet digital PCR front, Griffiths' lab hasn't yet received a RainDrop system, but it working with the company on several applications.

"The key areas that we're exploring are around free fetal DNA in maternal circulation for prenatal diagnosis," he said. "And then diagnostic hematology is another, and residual disease monitoring is the third."

For the free fetal DNA application, "we currently do Y chromosome detection for sex-linked disorders using a qPCR approach, and are exploring the droplet digital PCR as an alternative approach," Griffiths said, adding that one of his colleagues also just received a grant to extend this to prenatal diagnosis of single-gene disorders using free fetal DNA circulating in maternal blood.

"This is best delivered, we think, by droplet digital PCR on the RainDance … although this might also be targeted deep sequencing using NGS," he said. "We have to be flexible using these different platforms."

For diagnostic hematology, the group is exploring the RainDrop for detecting and quantifying low-level mutations. "The best example is the JAK2 V617F mutation in the myeloid proliferative neoplasms," Griffiths said. "Using essential thrombocytosis as an example, which is one of that group, some of the existing techniques used to diagnose this are not sensitive enough [to detect] the level of the [JAK2-V617F] clone. Therefore we need a technique that is both more sensitive than existing techniques, but also quantitative, because very soon there will be targeted treatments that will reduce the tumor burden, which we'll need to monitor, in the same way that we want to monitor BCR-ABL in [chronic myeloid leukemia]."

Segueing into BCR-ABL monitoring, Griffiths noted that his lab runs approximately 2,000 such tests per year based on RT-qPCR. However, "there is a problem in terms of standardization between different laboratories, and lots of work goes on trying to standardize this inter-laboratory variation," he said. A recently created international scale has helped matters, but "converting to that international scale has proved troublesome, because it's only recently that the standards have become available," he said.

"We think that droplet digital PCR could replace that because you get an absolute digital readout, which removes the need for standard curves … it could be much more reproducible between labs … it'll give better limits of detection in terms of sensitivity, and increase the accuracy … so all in all it could become a better approach," he concluded.

Meantime, Los Angeles-based DxTerity Diagnostics, which recently told PCR Insider it has developed a rapid assay for analyzing gene expression of as many as 100 genes directly from small amounts of formalin-fixed, paraffin-embedded clinical samples without prior RNA extraction, is also exploring using the RainDrop platform to enhance its test.

"We are interested in the technology’s ability to deliver higher multiplex, quantitative results," Bob Terbrueggen, president and CEO of DxTerity, told PCR Insider in an email.

DxTerity initially collaborated with RainDance to assess the compatibility of RainDrop with the assay, called the FFPE DxDirect Her2 assay, which the company hopes can improve the measurement of HER2 gene expression levels in FFPE tissue.

"This small study was designed to verify the ability to read our DxDirect assays using the RainDance system," Terbrueggen said. "Everything worked the first time out without any reagent modifications. We ran a two-color assay … and compared HER2 negative to HER2 [positive] FFPE samples. The fold change measured (32-fold) using the RainDance system was the same as observed by real-time PCR."

"Overall, we were pleased with the performance of the RainDance system and are intrigued by the opportunity that digital PCR offers for the development of simple, higher multiplex, quantitative gene expression assays," Terbrueggen said.

Jason Bielas, a scientist at the Fred Hutchinson Cancer Center, is currently eyeing a RainDrop platform to, as Watson previously alluded to, supplement the QX100 platform that it has already used to measure rare mutational events with frequencies of as small as one in 109 in nuclear and mitochondrial DNA.

"In the Bio-Rad system, in order to generate the droplets, you're required to use their chemistry, their polymerases, their master mixes," Bielas told PCR Insider. "I think one of the biggest advantages of the RainDance system is the fact that for the most part it's an open-chemistry platform … in which you can use a number of different polymerases and master mixes and still be able to generate droplets and do counts, as well, with TaqMan chemistry. That's what we're most interested in, especially since we'd like to use some of our own polymerases, maybe to do some long PCR, or even ligations in droplets. It just opens a broader range of applications for us in terms of different reactions we can perform in droplets."

More specifically, Bielas and colleagues a few years back developed some assays that can detect damage to DNA as opposed to mutations, and "in order to get those to run correctly we have to add specially engineered polymerases, master mixes, and vary the chemistry. They're really particular. In order to make them more robust, we'd like to use a digital [PCR] platform."

Bielas noted that his group has only been in discussions with RainDance about these ideas, and hasn't yet conducted experiments on its system.

Watson noted that about half of the potential customers for RainDrop are thinking about using it in "liquid biopsy"-type applications — in other words, detecting nucleic acids and, more specifically, mutated nucleic acids from the bloodstream of patients, either in cancer research or prenatal detection.

Other potential customers, the company said, include Ben Ho Park of Johns Hopkins University, who has been sending samples to RainDance for rare cancer allele detection using established TaqMan assays; Andy Brooks, an associate professor at Rutgers University and COO of the school's Cell and DNA Repository, who is investigating free fetal DNA in maternal circulating blood; a scientist at Beth Israel Deaconess Medical Center who is exploring multiplexed analysis of microRNA cDNA; and epigenetics company Zymo Research, which is conducting methylation analysis of biomarkers using minute starting quantities of material.

Application-wise, RainDance "started with a conservative focus [for RainDrop], looking mostly at rare allele detection," Watson said. "But more and more we have been seeing applications such as methylation detection, microRNA detection — anything that needs extremely precise quantitation."

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