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OHSU's Gordon Mills Prepping NanoString-Based Proteomic Platform for Clinical Work


NEW YORK (GenomeWeb) – Researchers from Oregon Health & Science University, MD Anderson Cancer Center, and NanoString Technologies have validated the company's nCounter platform for quantifying proteins in tumor cell lysates.

The work, published last week in Molecular & Cellular Proteomics, is part of a larger effort by OHSU researcher Gordon Mills to develop a cancer proteomics workflow suitable for clinical implementation.

The study demonstrates the "industrialization of the technology," said Mills, who is in the process of transitioning from his position at MD Anderson to a new position as head of precision oncology at OHSU's Knight Cancer Institute. "It is now at a scale and reproducibility where it really can be used [clinically]."

One of Mills' main initiatives at MD Anderson has been building his lab's Cancer Proteome Atlas, a collection of more than 8,000 tumor samples analyzed using reverse phase protein arrays. Many of these samples have come from the National Cancer Institute's Cancer Genome Atlas (TCGA) project, on which Mills' lab did a significant portion of the proteomic analyses.

By collecting and analyzing this large number of samples, Mills has been able to identify proteomic subtypes within various tumor types along with patterns of protein signaling that suggest particular targeted therapies. More recently, the project has shifted in a more clinically-focused direction, looking increasingly at questions such as how tumors change in response to treatment and the differences between primary tumors and metastases.

Mills' ultimate goal has been to use his lab's findings to guide patient therapy. To do this he decided to shift from RPPA to the nCounter system, which he said he believes is better suited to clinical work.

RPPA uses cell lysates spotted in array formats that can then be probed with antibodies to proteins of interest. RPPA assays can measure several hundred proteins in a sample of interest and are well suited to clinical cancer research due to their ability to work with small sample volumes.

However, Mills said, the technique is somewhat cumbersome, relatively low throughput, and provides only relative quantitation.

For clinical assays, "as compared to a research protocol, you really need something that is robust, reproducible, and deployable," Mill said. "And so I would say that in terms of moving to a CLIA laboratory, [based on] the ease of the [nCounter] technology and the reproducibility, this is ready to move into a CLIA environment."

Nanostring has sponsored portions of Mills' research and he has invented and licensed technology to the company.

NanoString's nCounter platform uses nucleic acid barcodes to allow highly multiplexed read-out of molecular targets. The company's work has traditionally focused on nucleic acid-based assays, but the platform can also measure proteins by using antibodies conjugated to the nucleic acid barcodes. This approach was first put forth by researchers at Massachusetts General Hospital in 2014, and the company and collaborators including Mills have worked to refine the approach since.

In the recent MCP study, the researchers established the platform's suitability for clinical work by testing its performance across technical and biological replicates with different users and different lots of antibodies and across independently run batches. They compared its performance to established antibody-based assays including Western blot, ELISA, and RPPA, finding good concordance with these methods.

They also used the system to analyze phosphoproteomic changes in response to treatment with drugs targeting PI3K and mTOR signaling and to classify a set of breast cancer patient tumors based on their proteomic profiles.

Mills said he and his Knight Cancer Center colleagues now aim to implement an initial clinical assay on the platform. That first assay will be a complement to and extension of NanoString's Prosignia breast cancer product, adding protein-level information to the test.

"We could do a single assay that does, for instance, HER2, proliferation, KI67, and all of the components of Prosignia to say, 'Here is everything you would ever want to know about this tumor,'" he said. "That's our current plan."

The initial stage of this project will focus on replacing individual immunohistochemistry assays currently done as part of a breast tumor workup with the more quantitative, multiplexed nCounter-based assays, Mills said.

He noted that while the nCounter-approach loses the spatial information provided by conventional IHC, it captures more of the tumor in its analysis, "which may give you a better representation that is less influenced by the heterogeneity of the tissue."

"I think that initially we will use it in cooperation with standard technology to ask how much more information content there is [in the nCounter assay]," he said. "Then, I think that with a little more validation in patient samples, we will be able to say that this is actually a very accurate representation of what is going on in the tissue. At least that's what the preliminary data in the paper shows."

That said, "It's going to take thousands of samples before we are going to be able to say this should replace IHC," Mills cautioned. "Basically, this [MCP study] is validation of the technology so it is ready for those types of assays."

Ultimately, Mills said, his goal is to have an nCounter-based assay that retains the spatial information provided by standard pathology approaches.

"We needed to show the robustness, the reproducibility of the technology in general," he said. "This is beautiful on lysates the way we've done it. It's as good as it gets. So, the next step will be to take that even further into a spatially-orientated platform."

"We're well along in that process," Mills added, saying he believed his lab was "about 80 percent of the way there." In 2016 NanoString announced the release of its Digital Spatial Profiling technology, which allows multiplexed measurements of up to 800 proteins and RNA while retaining spatial information.

"One of the challenges will be to increase the repertoire of antibodies that are usable, the number that can be analyzed at any one time," Mills said, "and then just to make the platform as reproducible as possible so that it can move to a CLIA environment in a spatially oriented manner."