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Exosome Surface Markers May Enable Cancer-Specific Dx Tests

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NEW YORK (GenomeWeb) – Though researchers and diagnostic development firms have for some time been exploring using exosomes — persistent, circulating vesicles that carry cellular nucleic acids and proteins — for cancer and other disease testing, the field has yet to see many tests translated into clinical use.

Much work has been focused on isolating the full spectrum of exosomes circulating in a blood sample, and then searching their RNA or DNA en masse for cancer biomarkers.

But recently, a few new methods have emerged that utilize a simpler strategy that could see a quicker route to the clinic — using surface markers to detect exosomes specific to a particular type of cancer and isolate them from the background of other circulating vesicles, cells, and cell-free nucleic acids.

Peregrine Pharmaceuticals recently reported on its own efforts in this area after licensing IP from the University of Texas Southwestern last year regarding the detection of exosomes that express the protein phosphatidylserine (PS).

The licensing agreement came out of a sponsored research agreement between Peregrine and UT that yielded preliminary evidence that cancer patients have higher levels of exosomes containing PS.

In the new study published in Oncotarget, the UT Southwestern investigators who developed the approach reported that they were in fact able to distinguish between healthy subjects and patients with ovarian tumors based on the levels of PS-positive exosomes in their plasma. 

According to the authors, tumor cells are known to express PS on their plasma membranes, and they hypothesized that in patients with cancer, there might also be PS-positive exosomes in the blood that could serve as a surrogate biomarker.

Peregrine CEO Steve King said in an interview that the company's attention to PS as a biomarker comes out of its therapeutics work.

"We have been studying for some time the fact that tumors express a phospholipid, or in other words they expose it, which is normally only present inside the cell," he said. "Since the concept of exosomes is that when tumors release them, they are mini versions of what the tumor looks like, surface-level PS appeared to be something that would discriminate tumor-derived exosomes" from other vesicles in circulation, he explained.

To detect exosomes bearing PS on their surfaces, the UT group used an engineered, multivalent PS-specific antibody to create a highly sensitive and quantitative ELISA for the detection of picogram amounts of PS in plasma, the authors wrote.

The team then analyzed blinded plasma samples from 34 patients with ovarian tumors and 10 healthy subjects for the presence of PS-positive exosomes.

Analyzing the results, they found that samples from patients with malignant ovarian cancer did indeed have significantly higher levels of PS-positive exosomes than those with benign tumors or healthy controls. Similarly, the malignant and benign groups both showed significantly higher PS exosome levels than seen in the healthy subjects.

"Interestingly, almost half the patients with benign disease had no detectable PS as did 100 percent of the individuals in the normal group," the authors wrote.

For malignant tumors versus normal controls the team calculated an AUC of 1.0 at an optimal cutoff point. For distinguishing benign tumors versus control samples, the AUC was 0.950, and for discerning malignant versus benign it was 0.911.

Though the results are only preliminary and need to be validated in independent samples, the researchers argued they suggest that the predictive accuracy of PS could be "generally excellent."

The new Peregrine data follows another study published in January by a team from Arizona State University, which showed that detection of cancer-associated exosomes sensitively discriminate patients with pancreatic cancer from both healthy controls and individuals with pancreatitis.

Although the Arizona team's approach also relies on targeting exosomes that carry a certain cancer-associated protein, it also involves novel advances for isolating biomarker-positive exosomes over the standard centrifugation and ELISA method used by Peregrine and its UT collaborators.

The Arizona researchers have developed a system to use the interaction between two nanoparticles to pull exosomes that express a particular molecular target on their surface from the larger pool of circulating bodies.

According to King, Peregrine doesn't anticipate trying to move its PS-based exosome test toward the clinic on its own. Instead, it is hoping to find a partner to commercialize the test.

Though the firm hasn't solidified any agreements yet, he said that one option might be a company that is already working on exosome-based diagnostics.

"We think this could be interesting to one of the companies that is already interested in exosomes, and that would give us the ability to work with them to identify these [tumor-specific] exosomes and then also be able to look inside to further characterize … and look for other markers that might help in treatment," King said.

One firm that could fit the bill is Exosome Diagnostics. So far, Exosome Dx has only developed tests that assay exosomal RNA. But the company recently launched a new product it calls Shahky, which could allow some of the same type of protein-based exosome detection assays as Peregrine and the Arizona team have published.

According to Exosome Dx, Shahky uses a label-free detection method, without upfront sample prep, to detect exosomal protein markers. The company hasn't described further details.

Aethlon Medical subsidiary Exosome Sciences also said last year that it had begun production of its ELLSA (enzyme-linked lectin specific assay) diagnostic platform, with plans to make it available to collaborators developing new exosome-based tests.

In the academic sphere, a group from Massachusetts General Hospital and Harvard Medical School has developed a platform for label-free, high-throughput, quantitative analysis of exosomes using transmission surface plasmon resonance through periodic nanohole arrays, work described in a publication in Nature in 2014.

More recently, the same researchers reported what they call an integrated magneto–electrochemical sensor for exosome analysis, which they said allows highly sensitive, cell-specific exosome detection.

A team from the University of Queensland shared its own approach for the quantification of clinically relevant exosomes isolated from patient serum, also using a surface plasmon resonance platform, in Scientific Reports last year. Similar to Hu and his colleagues, the Queensland team's method uses exosome surface markers CD9 and CD63, combined with tumor-specific markers.

Tony Hu, who led the ASU team in developing the nanoparticle-based pancreatic cancer exosome test, said when he and his colleagues released their results that as long as researchers can identify markers that delineate disease-associated exosomes from others, his team's nanoparticle-based platform should be able to read out a diagnostic signal.

For example, he and his colleagues showed that they could detect vesicles derived from tuberculosis bacteria in patient urine samples.

The marker that the Texas team used, EphA2 is also overexpressed in the early stages of colorectal cancer and non-small-cell lung cancer, in addition to pancreatic cancer, Hu said. Or if the goal were to detect exosomes or other vesicles with a specific cellular origin, the probe combinations of the assay could potentially be tweaked toward that end.

According to King, based on what Peregrine understands about phosphatidylserine so far, it's possible the approach the company has licensed from UT could also be applied to other cancers.

"All cancers share this common PS exposure, so we think that's a real advantage," King said.

Since licensing the approach from UT, Peregrine has been working on simplifying the assay to make it more amenable to wide clinical use. Using this "refined" version, the firm is now engaging with academic partners to potentially begin additional validation studies.

As a therapeutics company, Peregrine's initial interest in PS exosome detection centered around the potential for this type of a test to measure the efficacy of particular treatments. That type of longitudinal monitoring will hopefully be something it can continue to test as it moves forward, King said.

The UT researchers hinted at this potential, as well. In its study, the team was able to follow three patients longitudinally for about six months after surgery. Two showed no detectable PS in their plasma, while the third patient had significantly elevated PS suggestive of recurrence or residual disease.

When the group unblended these cases, clinical follow-up confirmed that the first two patients had no evidence of disease whilst the third patient did in fact recur.

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