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ASHG Studies Highlight Diverse Applications for Bio-Rad's ddPCR as Tech Moves Toward Clinical Use

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NEW YORK (GenomeWeb) – Researchers continue to develop applications for Bio-Rad's Droplet Digital PCR, and the firm last week highlighted three of the 18 presentations at the American Society of Human Genetics that described using the technology.

In an interview, representatives from the company also confirmed that Bio-Rad is in ongoing discussions with the US Food and Drug Administration to pursue 510(k) clearance for an oncology application using ddPCR.

Projects represented at the ASHG meeting, held last week in Baltimore, included work on copy number variations, mosaic analysis, oncology and liquid biopsy, as well as genome editing methodology.

The ddPCR technology has amassed a number of studies, and the recent work presented at ASHG fits into overarching applications spaces, showing "a wide arc in terms of utility," George Karlin-Neumann, director of scientific affairs at Bio-Rad's Digital Biology Center, told GenomeWeb in an interview this week.

Bio-Rad's Jennifer Berman, a scientist with the firm, told GenomeWeb that one of the studies presented at the conference highlighted a trend for using ddPCR to build on discoveries made in broader surveys.

Specifically, the lab of Diptasri Mandal at Louisiana State University looked at germline copy number variations that could be associated with hereditary prostate cancer (HPC), particularly within the African American community.

They used array comparative genomic hybridization to discover regions duplicated in a small set of HPC cases analyzed.

The work demonstrated how ddPCR can be a complementary follow-up tool as people are making their discoveries with global tools like array-CGH, particularly if they want a method "to both validate those and follow through on them in a lower-plex, lower-cost way, but also have the high precision and sensitivity," Berman said.

"It illustrates researchers starting with a global tool — some might do NGS, some might do arrays — and then, when they identify regions of interest, they go for an orthogonal method to validate it, but also a bit more of a nimble method for a small-plex study," she added.

Karlin-Neumann noted that this strategy has even resulted in the creation of new companies.

Hanlee Ji, whom GenomeWeb interviewed in 2012, used NGS methods to identify drivers of hereditary gastric cancer. These were then made into a CLIA lab test using ddPCR, which was the basis for founding a company, Toma Biosciences.  

Other studies presented at the meeting dealt with somatic mosaicism, using ddPCR to look for either mutations or copy number changes in a fraction of cells being assayed.

The lab of Fred Gage at the Salk Institute, which studies stem cells in the hippocampus of adult brains, revealed data suggesting endogenous retrotransposons generate somatic mosaicism in neuronal and non-neuronal cells. "They were using ddPCR to validate some of the line-element mosaicism they were observing by NGS in single cells," Berman explained.

There was also work presented on mosaicism in sporadic Parkinson's disease, and "In general, there continues to be a community who sees the utility of ddPCR for assessing somatic mosaicism," Berman noted.

Finally, Bio-Rad has begun developing methodology to use ddPCR for detection and validation of genome editing events, as recently described by GenomeWeb.

An ASHG poster presented by Berman described work done in collaboration with Bruce Conklin's lab at Gladstone institute at University of California, San Francisco.

"Their endgame is to edit stem cells for studying cardiomyopathies and other diseases," Berman said. "They're heavily involved in optimizing editing tools to achieve those goals."

However, editing introduces both homology-directed repair (HDR) and non-homologous end joining (NHEJ).

"HDR is essentially the precise edits ... and NHEJ are insertions or deletions that are a bit more haphazard and are considered collateral damage," Berman said.

The poster described an assay using ddPCR to quantify both HDR and NHEJ outcomes of genome editing in a single well, allowing researchers to screen different genome editing methodologies and assess which conditions yield more HDR versus NHEJ.

Berman said the firm has developed rapid screening for simultaneous, single-well quantification of HDR and NHEJ, as well as more "garden variety" assays for detecting each type of event separately.

"This is faster and more sensitive in some cases than an NGS readout, and definitely more cost efficient" Berman noted.

Bio-Rad also had two guest speakers present at a lunch session. Both were researchers in the cancer space, and they highlighted the use of ddPCR in liquid biopsy assessment, examining tumor mutations from circulating cell-free DNA. The firm has customers using ddPCR for mutation detection from cfDNA, to look at tumor heterogeneity, and in the rare cancer mutation detection space, Berman said.

"A lot of the posters highlighted here we see in the copy number space, but actually the mutation detection application space is quite large for us, in particular in cancer," she said.

A conference called IDWeek was also happening at the same time as ASHG, and Berman noted that there is an enthusiastic community pursuing ddPCR for detection and quantification of viruses and in microbiology.

There is also a growing number of researchers using ddPCR for epigenetics studies, Karlin-Neumann said, pointing to recent work in oncology to predict head and neck cancer recurrence, as described by GenomeWeb. Meanwhile, another group developed a ddPCR-based technique called MethyLight, also recently covered by GenomeWeb, and still another has been using ddPCR to assess methylation status in diabetes.

Many of these are research applications, but clinical use of the technology may be imminent.

Bio-Rad is currently pursuing 510(k) clearance for its platform. "We are in discussions with the FDA about getting the system cleared and a specific assay approved as well," Karlin-Neumann said.

In parallel there are labs using the technology for CRO work to support clinical trials as well as for patient use, he said.

Karlin-Neumann noted there are a few active studies using ddPCR listed on ClinicalTrials.gov. In one, patients with EGFR mutations in plasma detected by droplet digital PCR will receive gefitinib, while in another ddPCR will be used to detect biomarkers of spinal muscular atrophy in infants, for example.

"We know from that, and from conversations with users, that people are trying to advance [the technology] beyond proof-of-principle studies into larger prospective trials," he said.

In addition to Toma's CLIA service, Biodesix announced early this year that it will launch a ddPCR-based test for BRAF, KRAS, and EGFR in the plasma of lung cancer patients.

The Bio-Rad test that will be submitted to FDA is an in vitro diagnostic oncology test, and Berman described it as "an active effort."

"At the same time there are parallel efforts to get it approved and cleared in Europe under CE-IVD," Karlin-Neumann added. 

Bio-Rad's competitor, RainDance Technologies, has also described an industry-wide intent to clear platforms with the FDA, as previously reported

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