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SickKids Team Using NanoString Platform to Develop Gene Fusion Assay for Sarcoma Patients

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SAN FRANCISCO (GenomeWeb) – Researchers from the Hospital for Sick Children in Toronto are using NanoString Technologies' nCounter molecular barcoding platform to develop a clinical assay to detect gene fusions in sarcoma patients with the aim of providing the most accurate diagnoses and treatments possible.

At the Cambridge Healthtech Institute Molecular Medicine Tri-Conference here yesterday, Gino Somers, head of pathology at SickKids and an associate professor at the University of Toronto, said in a Nanostring-sponsored presentation that he and his colleagues are currently comparing their nCounter assay and "gold-standard" RT-PCR assays to retrospectively analyze sarcoma patient samples.

In addition, the researchers plan to begin a similar prospective analysis over the next 12 to 18 months, while others at SickKids are using the nCounter to develop a multi-gene diagnostic panel for medulloblastoma and to explore microbiology applications, Somers said.

"I think [nCounter] is very close to becoming a very useful clinical diagnostic tool," Somers said during his talk.

NanoString's core technology uses colored barcodes –- each of which is attached to a probe corresponding to a gene of interest – and single-molecule imaging to count hundreds of transcripts in one hybridization reaction. Advantages of the platform include multiplexing of up to 800 targets per reaction and direct hybridization, which eliminates issues that may arise in multiplexed PCR.

The company is taking a three-pronged approach to the molecular diagnostics market, Jay Gerlach, senior marketing manager for clinical markets at NanoString, noted in his introduction of Somers. For several years it has offered commercial "CodeSet" kits to profile gene and microRNA expression, and to analyze copy number variation in samples using its nCounter analyzer.

The company also develops its own molecular diagnostic assays, such as the Prosigna Breast Cancer Prognostic Gene Signature Assay, which received 510(k) clearance from the US Food and Drug Administration in September 2013.

But the company has also been attempting to gain a foothold in the laboratory-developed test market with its nCounter Elements – essentially assay reagents based on the barcoding technology that customers can use to develop their own assays of interest.

This is the focus of Somers' lab at SickKids, where researchers have been using NanoString's technology since at least 2010.

Somers' research focuses on sarcomas, tumors of connective tissue that disproportionately affect children and require very accurate diagnoses due to subtle differences in the way the tumors behave and respond to treatment.

Depending on specific gene fusions that are present, sarcomas, and Ewing's sarcomas in particular, "can be very aggressive, or some can be indolent," Somers told GenomeWeb after his talk.

Accurately identifying these myriad gene fusions, however, is a difficult task, with FISH and RT-PCR currently serving as the standard methods. However, the NanoString platform is poised to become the next gold standard, Somers noted.

One of the major advantages of the nCounter platform is that it requires only 200 nanograms of input RNA, whereas FISH and RT-PCR tests can require much more. "We are getting very small biopsies and sometimes we only have enough RNA to run one or two PCR tests," Somers said. In addition, nCounter's high degree of multiplexing and lack of a reverse transcription step help save time and money.

Somers' lab has designed nCounter Elements probes to cross the breakpoints of the various fusions that drive sarcomas, so the assay can identify which pair of genes are involved in the fusion. During his talk he presented some high-level results from an initial study in which his lab used the reagents to detect 14 specific fusion transcripts using a variety of sample types, including fresh frozen, formalin-fixed paraffin-embedded, and tumor crude extracts.

In their tests, which required only 200 nanograms of sample RNA or 400 nanograms of crude tumor extract — compared to up to 2 micrograms for RT-PCR — the nCounter assays were able to distinguish, for example, between two types of nearly identical Ewing's sarcomas with slightly different fusions.

Somers and his colleagues were experiencing some cross-hybridization in certain samples, however, so they went back to the drawing board to develop a second iteration of their probes with an improved design. They have used these new and improved probes on 11 patient samples, and the assays are "very clean … [and] allow us to tell the transcripts apart and test for multiple fusion transcripts in the same assay," Somers said. However, he noted, results from this second go-around are only a few weeks old, so he was not at liberty to share data in great detail.

The group also found that nCounter assays performed just as well using crude tumor extracts as they did using RNA from frozen tissue. This "may cut out 24 to 48 hours of extraction time" in the lab, which is "very promising," Somers said.

One audience member at Somers' presentation pointed out that RNA sequencing can accomplish the same thing as the nCounter assays, and wondered what the advantage of the latter would be.

"I think RNA-seq is a very powerful tool with a role in clinical diagnostics," Somers said. However, "cost is still an issue," and the amount of data that is produced is a little like "using a sledgehammer to kill a fly," he added. NanoString's platform also has a much shorter turnaround time and produces "very clean results."

Lastly, he highlighted the versatility of the nCounter platform, noting that a SickKids colleague is currently developing a medulloblastoma panel on the platform, and that still others are exploring outside the realm of cancer in the area of microbiology.

This latter application is being explored "anywhere you'd use a PCR assay," he told GenomeWeb after his talk. For example, one of his colleagues is exploring the platform for subtyping of human papillomavirus.

Nanostring's Gerlach told GenomeWeb that the company is aware of "lots of interest from customers" in microbiology applications for nCounter; however, it is not an active area of R&D for the company, which is keeping its internal focus on cancer applications.

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