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Researchers Debut Method for Combined Separation, Gene Expression Analysis of CTCs

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NEW YORK (GenomeWeb) – Platforms and methods for capturing rare cells and probing them for molecular insights have proliferated in recent years, but generally the two steps have remained distinct.

Now a research team from the University of Toronto has developed a single approach that enables both isolation of CTCs and and analysis of their gene expression, or messenger RNA content.

In a study published this week in Nature Chemistry, the researchers tested the method, called single-cell mRNA cytometry, first on cell lines, and then in a small number samples from patients with prostate cancer, hinting at potential clinical applications in oncology.

Shana Kelley, the team's lead investigator, said that the approach reflects an attempt to "kill two birds with one stone."

"We can go after specific sequences that we use for capture, which also tell you something about the patient," she explained.

The method uses two classes of magnetic particles that are labelled to selectively hybridize with different regions of a target mRNA. Hybridization of these probes to the molecule in question causes the formation of large magnetic clusters inside a cell of interest. In the study this was a circulating tumor cell, but the method could also be applied to other rare cells by choosing some other relevant target mRNA.

The magnetic clusters allow the cells to be separated out and also provide a way to assess the amount of RNA, or the level of gene expression, in each cell without the PCR amplification commonly used in similar single-target analyses.

First, the investigators tested the method on various cell lines. Then, to provide a hint at potential clinical utility, they picked two targets — TMPRSS2/ERG and AR-V7 — to search for in blood samples collected from a small cohort of patients who were undergoing treatment for metastatic castration-resistant prostate cancer.

For each of a handful of patients, the group identified CTCs in parallel using immunofluorescence and either their mRNA method or an EpCAM-based capture, concluding that the mRNA cytology method appears to be able to provide both a CTC count and an accurate readout of the absence or presence of clinically relevant mRNAs.

Kelly explained that the potential utility of the approach would be in areas where clinical researchers currently combine some sort of label-based isolation of CTCs with a follow-on analysis of mRNA levels, usually involving a PCR reaction.

The fact that the team chose to look at AR-V7 in the study is relevant, considering the fact that the clinical field is just now seeing the first commercialized tests that use AR-V7 levels to help guide treatment decisions for men with advanced prostate cancer.

The first commercial test in this area is marketed by Genomic Health and uses a CTC analysis method developed by Epic Sciences, which does not isolate CTCs, but rather spreads out all of the cells in a blood sample, and uses microscopy and imaging techniques to locate the CTCs and assess protein expression in their nuclei.

But a group from Johns Hopkins has also developed an mRNA-based approach, using an antibody-based capture and RT-PCR method called the AdnaTest. Qiagen, which acquired the AdnaTest technology several years ago, has licensed IP from the Hopkins team and is planning to commercialize a research use only kit in the near future.

Kelley said her team's mRNA cytometry method offers an alternative to combined approaches like the one used in the Qiagen AdnaTest.

"Our performance is really good relative to marker-dependent methods," she said. "We get very high efficiencies in spiked samples where we are capturing both higher EPCAM and lower EPCAM cells."

The method might be less likely to compete as directly with label-free CTC capture methods — like Vortex Biosciences' VTX-1, Angle's Parsortix, or the CTC iChip — which rely on fluidic separation and concentration of CTCs based on their size, shape, or malleability, pulling out a larger population of cells for analysis, regardless of whether they carry a particular genomic target.

"It's all about what are you looking for in a sample," Kelley said. "What we think we can do well with this is make targeted measurements much, much simpler, where you want to know 'Are these cells positive for the marker I am targeting, how many of them are there, and what is the expression level?'"

Kelley and her coauthors wrote that one important next step will be to test the method on earlier-stage cancer patients, whose samples typically exhibit much lower numbers of CTCs.

She said the group is also looking to apply it to other disease areas, and in stem cell research where it could be useful in isolating cell subpopulations or in quality control.

As far as AR-V7 is concerned, Kelley and her team haven't had conversations yet about applying mRNA cytometry in commercial test development, but are hoping that there will be interest from others in using it.

"It's an easy measurement and it's actually a cheap measurement relative to other types of workup that can be applied," she added. "And with these markers, as we really understand how they can be applied, we need to get away from research-grade type of approaches. We need things that can be used in labs straightforwardly, and I think this technology is a great fit for that. It lends itself to a very straightforward implementation if we were to try to make a lab-grade instrument."

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