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Danish Study Backs Sensitivity of Exiqon qPCR Panels for Detecting miRNAs in Blood


By Ben Butkus

Exiqon's qPCR-based microRNA-quantification panels are more sensitive than competing qPCR- and microarray-based miRNA-quantification assays, particularly with the extremely low miRNA levels typically found in blood plasma or serum, according to a recently published independent research study.

The results add to a growing body of evidence that Exiqon's microRNA panels – currently for research use only – hold promise as a blood-based diagnostic or prognostic tool for cancers characterized by miRNA markers in the bloodstream.

To that end, the company is progressing in its previously announced effort to develop a blood-based diagnostic for early-stage colorectal cancer, and by the end of the year plans to have screened around 1,000 individuals in various validation studies with an eye toward a subsequent prospective clinical trial, company officials said this week.

Exiqon has also identified miRNA signatures that it believes can help diagnose melanoma and distinguish cutaneous T-cell lymphoma from benign, chronic skin conditions, and is in the early stages of developing qPCR panel-based tests for those indications.

In the most recent research study, published last week in BMC Genomics, scientists from Aarhus University Hospital in Denmark – longtime collaborators of Exiqon – set out to evaluate the performance of Exiqon's miRcury Ready-to-Use PCR Human miRNA panels I+II V1.M, part of the company's miRcury LNA Universal RT microRNAPCR product line.

More specifically, the Aarhus researchers compared the ability of Exiqon's miRNA panels with two other commonly used commercial miRNA-quantification panels — Affymetrix's GeneChip miRNA 2.0 array and Life Technologies' Applied Biosystems TaqMan Human MicroRNA Array v3.0 — to quantify different ratios of 174 synthetic target miRNAs spiked into both synthetic microRNA and human plasma RNA samples.

The researchers focused in particular on the performance of the platforms using extremely small levels of RNA input such as what can be extracted from, for example, just 250 µL of human plasma, in order to better approximate clinical conditions.

They found that the GeneChip miRNA 2.0 platform was not sensitive enough to reliably produce signals with the plasma RNA input levels studied, and this eliminated that assay from further assessment.

Meantime, the researchers found that both the Exiqon and Applied Biosystems assay panels had comparable high levels of sensitivity in samples with abundant miRNA levels. However, at low miRNA levels the sensitivity of the MiRcury platform was significantly higher than the TaqMan platform, the researchers reported. At the very lowest levels of miRNA – the equivalent of 0.5 to 2 template copies per amplification reaction – neither of the platforms were senstive enough to reproducibly detect the targets.

The researchers speculated that the sensitivity differences between the platforms could be attributed to several factors. These include differences in the cDNA synthesis used by each platform: "MiRcury uses a universal approach with poly(A) end-tailing and oligo(dT)-primed reverse transcription," the researchers wrote, that may be more robust and sensitive than the TaqMan approach, which "megaplexes [more than] 300 miRNA-specific stem-loop primers for initiation of reverse transcription."

The authors also hypothesized that the inclusion of Exiqon's locked nucleic acid, or LNA, technology in the MiRcury primer designs may benefit Exiqon's assay. "LNAs make assay design nearly independent of miRNA GC content and compensate for many of the compromises one otherwise would have to make with a short miRNA target sequence of just [approximately] 22 nucleotides," they wrote, noting that LNAs have previously been reported to improve PCR sensitivity.

"I think the study shows that we have a robust system with great performance and sensitivity in very important types of samples such as plasma," Henrik Pfundheller, senior vice president of sales and marketing at Exiqon, told PCR Insider. "We believe the platform is well-suited to be used in a diagnostic setting."

Adam Baker, director of diagnostic product development at Exiqon, added that the study "is a really good independent qualification … that the PCR system works really well in this blood serum, which is what we've been promoting. It's nice to see that other people are now agreeing with us that this is a great system for that."

One major caveat to the Aarhus study was that Exiqon's assays produced a significant amount of false positives in samples lacking target miRNAs, raising questions about the technology's specificity. Meantime, the TaqMan platform generally showed no false positives in the control samples.

"However, our analyses of the synthetic samples revealed that within the operative range, i.e. from the first detection to the detection threshold, the two platforms produce nearly identical numbers and rates of false positives," the researchers wrote. "Hence, for practical purposes the specificity of the two platforms appears equal."

The Aarhus researchers also noted that the while the specificity issues of qPCR approaches may not ever be completely eliminated, it may be a moot point for miRNA detection and quantification due to the "ongoing technological shift" to alternative approaches such as small RNA sequencing, which "has the potential to replace qRT-PCR as the preferred tool for profiling low-abundance miRNA samples."

Nevertheless, Exiqon continues to move forward with diagnostic development using its MiRcury LNA miRNA profiling platform. Baker told PCR Insider that for the blood-based colorectal cancer early-detection program, which the company has been working on for more than a year (PCR Insider, 9/30/2010), "we're currently doing quite large-scale multiple validation studies of the signature … we discovered in 200 samples. We're going to be updating that at the end of the year … and we want to have screened 1,000 patients."

In addition, the company "now has access to a prospective clinical trial in Denmark" once the validation studies are completed, "and we are always looking for partners to test this in other ethnic groups, such as in the US," Baker said.

Beyond colorectal cancer, Exiqon has also identified promising diagnostic biomarkers of melanoma; and of cutaneous T-cell lymphoma, in order to distinguish between the malignant disease and chronic diseases such as psoriasis, atopic dermatitis, and eczema. An early study related to the latter program, conducted in collaboration with the University of Copenhagen, was published two weeks ago in the journal Blood.

Have topics you'd like to see covered in PCR Insider? Contact the editor at bbutkus [at] genomeweb [.] com.

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