NEW YORK – With the launch of its highly multiplexed digital PCR platform, the Naica Prism 6, at the American Society of Human Genetics annual meeting this week, Stilla Technologies is forecasting a new horizon for digital PCR.
At a company-sponsored presentation last week preceding the ASHG meeting, researchers and clinical diagnostics developers demonstrated novel liquid biopsy applications on the Prism 6, including a 24-plex digital PCR cancer panel, as well as enhancements to basic science research afforded by the system.
Digital PCR yields absolute quantification and single-molecule sensitivity for target DNA or RNA within a sample, without the requirement for standard curves needed in qPCR. The Naica Prism 6 system also includes six fluorescence channels and can be used with chemistries enabling detection of additional targets in each channel, with as many as 24 distinct targets in a single assay now demonstrated.
In an interview, Stilla CEO Philippe Mourere said the firm expects highly multiplexed digital PCR to fit unique niches in oncology, infectious disease, and wastewater testing, as well as genomics research and other markets.
Mourere joined Stilla about 10 months ago after 25 years in executive positions at life sciences firms. Stilla’s six-color system was first described in 2019, initially made available for some users last year, and subsequently offered as part of a formal early-access program in June this year. At ASHG, Stilla is announcing the full global commercial availability of the platform.
The Naica Prism 6 system uses the firm's crystal digital technology, whereby approximately 30,000 individual PCR reaction droplets are aggregated into a "crystal" and imaged for each of the desired targets. Users have access to the raw images and are provided software called Crystal Miner to help interpret the matrix of results.
Overall, the system can absolutely quantify many targets in a sample within a matter of hours, Mourere said.
As various presenters showed at the ASHG event, this can be particularly useful in cancer research, as mutation signatures evolve over time and can differ between tumor and circulating tumor cells or cell-free tumor DNA. A highly multiplexed test can detect this signature using small samples and provide the status of each of the possible mutations in the context of one another, he said.
Lao Saal, chief operating officer at Swedish liquid biopsy outfit Saga Diagnostics, presented an assay at the ASHG industry talk that detected 24 different mutations in the PIK3CA gene using proprietary dPCR chemistries, called SAGAsafe. Saga Diagnostics provides testing services as well as research-use and CE-marked kits from its lab in Lund.
SAGAsafe can improve digital PCR’s limit of detection by "about 100-fold," Saal said, namely by eliminating false-positive droplets. "SAGAsafe is compatible with all of the digital PCR machines we've tested so far — Bio-Rad, Qiagen, the Combinati/Thermo Fisher box, and now with Stilla," Saal said.
The SAGA team deployed its 24-plex PIK3CA assay in a small cohort of breast cancer patient samples for which it also had next-generation sequencing data and found good minor allele frequency concordance between NGS and the Stilla Prism, Saal said.
With the 24-plex test, the team also saw a limit of blank in the 0.003 percent range or below, with limits of detection just under 0.01 percent.
"We are very happy with these results, the quality of the data, and the performance of the six-color Prism machine," Saal said. His team had only been working with the Prism 6 system for about three months, he said, but it intends to use it for more assays going forward.
Evi Lianidou from the University of Athens in Greece also presented clinical oncology research, tracking tumor evolution through the detection of low-abundance mutations in cell-free DNA and circulating tumor cells.
Lianidou and her team developed an assay on the Naica Prism 6 to directly compare eight cancer-related mutations in the EGFR gene — specifically exon 19 deletion, exon 20 insertion, and the single nucleotide polymorphisms T790M, C797S, L858R, L861Q, G719X, and S768I.
In a cohort of 48 non-small cell lung cancer patients, the team compared results within and between patients, looking at the presence of mutations in primary tumor biopsies, plasma cell-free DNA, and circulating tumor cell-derived genomic DNA. They also examined these three sample types at two different time points — before and after treatment with osimertinib (AstraZeneca’s Tagrisso) — for all eight markers, to begin to understand mechanisms of resistance to therapy.
As published earlier this year in Cancers, the team found a tremendous variation between samples, sample types, and time points.
The researchers also showed that for cfDNA specifically, the Stilla dPCR system was highly concordant with the US Food and Drug Administration-cleared Roche Cobas EGFR Mutation Test v2, which detects a total of 42 targets. Lianidou noted as well that her lab is accredited according to the ISO-15189 standard for EGFR testing in plasma samples with the Cobas technology.
"We see a very high concordance — more than 90 percent in all cases for each mutation detected," Lianidou said in her presentation. "Moreover, we have seen a number of cases where EGFR T790M was detected only by droplet digital PCR, and not by the FDA-cleared assay from Roche."
In the genomics research space, German Gornalusse at the University of Washington's department of obstetrics and gynecology has found numerous applications for the Naica Prism 6, including in sexually transmitted disease research like HIV genotyping and reservoir quantification, antiretroviral therapy response, and chlamydia testing.
In his ASHG presentation, Gornalusse delved more deeply into a National Institutes of Health-funded project querying the heritability of addiction. Preliminary data gathered from semen samples donated by heroin users in Seattle showed alterations in tRNA fragments, otherwise known as tRFs, which are thought to play a role in transgenerational epigenetic inheritance.
The Naica system correlated well with NGS data and qPCR assays, he said. The team used its preliminary data from the Stilla system to win funding from the Center for Aids Research, and has since applied the award to purchase the Prism 6, Gornalusse said. He intends to measure concentration and cleavage patterns of four tRFs in opioid users before and after treatment with suboxone, while he and his UW colleagues will also use the system for HIV reservoir monitoring and other applications.
The clinical 'sweet spot'
For clinical applications in particular, a quick turnaround of accurate, actionable data is the holy grail.
In Saga's case, the company is "trying to democratize cancer liquid biopsy testing and have it widely used in clinics," Saal said. "We see digital PCR being a major part of that democratization," he added.
"There is a need for having more targets in [an oncology] test — NGS can do hundreds but that is probably overkill. Having a test that can do a dozen, or several dozen, is a real sweet spot for digital PCR in clinics," Saal said.
The two technologies will still likely have a lot of interplay, however, and "there's lots of room for both to coexist," Saal said, describing a cancer use case involving an upfront NGS screen or chromosomal rearrangement detection followed by use of dPCR for patient monitoring.
Saga committed to digital PCR 10 years ago due to the technology's ability to provide direct readouts without standard curves, which enhanced comparisons between hospitals and labs. The firm has grown to a staff of 17 and recently brought in Peter Collins — formerly of Inivata, Guardant Health, Yourgene, and GSK.
The team has used "pretty much all" of the commercially available dPCR systems, Saal said. "We like the Stilla box a lot — it is difficult to compare and contrast them all, but in Stilla with six colors, that is as good as it gets," he said.
Stilla's Mourere said that for clinical tests that benefit patients, the firm realized early on that market demand was evolving "very rapidly" from single genes or a small number of targets to a broader signature.
"We strongly believe that the market is now ready to adopt a high-plex digital PCR modality, to bring the sequencing research ... from the discovery market into actionable signatures and biomarkers in regulated markets," Mourere said, adding, "We had to unlock a certain level of 'plex' to call it a signature."
At the ASHG presentation, Stilla's chief technology officer, Rémi Dangla, also noted that the field has been expanding dramatically, with more than 4,300 publications so far in 2021. Digital PCR is "being used in all fields of molecular biology, with the main driving fields being oncology and infectious disease," Dangla said, adding that there are also applications for environmental and food testing.
Growth in the digital PCR space of late — including Thermo Fisher Scientific's acquisition of Combinati last month — is good for the market overall, Mourere said.
"We are super happy to see that powerful organizations are continuously investing in digital PCR. It only further validates our position," Mourere said, highlighting that Thermo's investment occurred before Combinati even had any revenue.
Nevertheless, Mourere said he believes Stilla's crystal PCR is sufficiently differentiated to serve the translational research and clinical markets.
"What we are demonstrating today [with the Prism 6] is we are innovating yet again, before anyone else, setting a much higher bar on the market," he said.
Digital PCR technology, overall, has seen uptake as a research tool and in applied markets, such as wastewater testing, but the diagnostics space is a sweet spot.
"With the expansion of our level of plex, we are basically opening up new markets beyond those we have already been playing in," Mourere said. "We are continuing marching on downstream of translational research into the IVD space," he said, adding that the firm will be publicly announcing more details in the coming weeks.
The digital PCR space has seen its fair share of intellectual property lawsuits. Mourere noted that Stilla's recent settlement with Bio-Rad was an important step for the space and for the company itself, as it provided clarification to the market and signaled that the platform can be incorporated into regulated, companion diagnostic, and other workflows.
Saga's Saal said his firm's pharmaceutical business can benefit in particular from digital PCR, as more sensitive assays can yield more patients qualified for trials.
As an example, he showed analysis of samples from 204 breast cancer patients querying three hotspots in the PIK3CA gene. Using 600X NGS, 60 patients were positive for mutations, while using the team's SAGAsafe chemistry, 107 patients were detected. This is important, as PIK3CA mutation-containing breast cancers can be targeted with alpelisib (Novartis' Piqray), a chemotherapeutic drug that was cleared in 2019 by the US Food and Drug Administration.
Saga's team was also able to transfer its PIK3CA assay to the multiplexed format quickly, which Saal said was due in part to the fact that "the Stilla box is pretty easy to work with." He suggested Saga's other assays could also be ported to a highly multiplexed format with minimal optimization.
"Six dimensions opens up a new way of thinking about the data," he said, adding that the team has only just begun to conceive of applications for the system.
For detecting rare targets in tiny samples from patients who may have precious little time left, the benefits of high multiplexing are clear. In their Naica Prism 6 analysis of CTCs, for example, Lianidou's lab enriched using the Parsortix technology from Angle, subjected the resulting genomic DNA to whole-genome amplification, then used 15 microliters of amplified DNA in a 1 to 100 dilution in the Naica multiplex test.
The detection of complementary information from CTCs and cfDNA may have clinical utility, but to be used in the clinic the detection technologies must be well-validated and reliable with small samples, Lianidou said. "I strongly believe that multiplexing and using a very sensitive technology, like digital PCR, will allow us to do this," she said.
In terms of assays for the Prism 6, Mourere said content is very important to Stilla, and that improving its value proposition with content is an axis of continued development.
"The expansion from RUO into a regulated space is also part of our strategy — we are going to signal to the market that we have been very active in diagnostics," he said.