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Extracellular Vesicles Show Promise as Early-Stage Liver Cancer Diagnostic


NEW YORK – Researchers at the University of California, Los Angeles have developed an extracellular vesicle-based surface protein assay to detect early-stage hepatocellular carcinoma (HCC).

The work adds to the growing interest in using extracellular vesicles (EVs) for early cancer detection, underscored by a proliferation of companies exploring the space, including one started by one of the UCLA researchers.

HCC accounts for between 80 and 85 percent of primary liver cancers in the US and is often accompanied by a poor prognosis due to its generally advanced stage at the time of diagnosis.

In a clinical study published in the journal Hepatology, UCLA's Hsian-Rong Tseng, Yazhen Zhu, and their colleagues described a method for diagnosing early-stage HCC by identifying and quantifying tumor-associated markers on EVs, establishing a score for HCC presence and discriminating it from diagnostically similar conditions such as liver cirrhosis.

Tumor burden is generally very small in early stages of HCC and other cancers, explained Zhu. Accordingly, these tumors release limited amounts of genetic material into circulation, which can impact the effectiveness of liquid biopsy at these stages.

In contrast, EVs are much more abundant, being naturally shed by all cells and even more so by cancer cells. These can be targeted by directing antibodies against various surface markers, some of which are more cancer-specific.

"In a nutshell, our technology targeted tumor-derived EVs or subpopulations of EVs," Zhu said. "Then we can just avoid the huge amount of background EVs."

Zhu and colleagues' method uses a technology called click beads to purify EVs by capturing biomolecules through a rapid conjugation reaction, which they quantify using duplex real-time immune-PCR. This allowed them to develop the HCC EV ECG Score, where ECG stands for the three main EV subpopulations defined by the surface markers EpCAM+ and CD63+; CD147+ and CD63+; and GPC3+ and CD63+.

To optimize the technique for discriminating HCC from liver cirrhosis, they conducted a Phase II case-control study involving 45 treatment-naïve, early-stage HCC patients, 23 patients with other cancers, and 61 patients with liver cirrhosis.

The UCLA researchers assessed the score's utility as a surveillance test by restricting the patients with HCC to Barcelona clinic liver cancer stages 0-A and the controls to at-risk cirrhosis. Participants with the other cancers were used as controls to confirm the test's specificity toward HCC.

The three ECG EV subpopulations were highly associated with HCC diagnosis and could distinguish HCC from cirrhosis. Furthermore, they demonstrated 90 percent sensitivity for identifying tumors within Milan criteria, a measure for determining whether HCC patients may move forward with liver transplantation, emphasizing the technique's potential as a means for detecting cancers while still curable.

The investigators aim to carry their EV biomarker score through to Phase III clinical testing. But to do it right, Tseng says that they will first "go back to the drawing board" and further optimize their method, as they have since developed "several ways" to further lower background noise and increase signal.

"We will [also] increase the sizes of training and validation cohorts by incorporating plasma samples from other institutions," Tseng said.

To that end, Tseng's group is working with Amit Singal, a hepatologist at the University of Texas Southwestern Medical Center, who has accumulated a large library of HCC patient plasma samples.

"We just finished our material transfer agreement," Tseng said. "Once we lock down our experimental parameters, we will start to do double-blind testing on his plasma samples."

Institutions participating in the study include UT Southwestern, UCLA, and Cedars-Sinai Medical Center. The group has so far enrolled patients at UCLA and Cedars-Sinai and plans to expand to several East Coast institutions in the near future.

Although Tseng and his colleagues are not yet working on licensing their HCC EV assay, Tseng mentioned that they may look to do so later with his own company, Eximius Diagnostics.

"Eximius Diagnostics will possibly carry this IP forward," he said.

Founded in 2020, Eximius is based around the EV Click Chip, a custom microfluidic chip that captures EVs in a two-step process mediated by tetrazine antibody-grafted silicon nanowire substrates and a network of microchannels altered to induce chaotic mixing.

For the current study, however, the group switched to a bead-based capture system, which Tseng described as comparatively simpler than the chip.

"The beads technology is very compatible with most laboratories' operations," he said, requiring only common items to implement, such as pipettes, filters, and a PCR machine.

Eximius is currently wrapping up an entry round of financing, which it expects to close around the end of the month, after which the company plans to initiate a Series A round of fundraising.

"We're looking into a very aggressive timeline for Series A, which will be in the summer of 2023," Tseng said.

While HCC remains the UCLA team's primary focus, the researchers are beginning to investigate the diagnostic potential of EVs, as well as their click chemistry-based technology, in other diseases.

"We have a pipeline [where] we are trying similar technology in prostate cancer, thyroid cancer, and even lung cancer," Zhu said, adding that they are also exploring applications in the noninvasive prenatal testing field.

Last year, for instance, Zhu, Tseng, and colleagues published a study in which they adapted the EV Click Chip to target circulating trophoblasts in order to screen for placenta accreta spectrum, a high-risk obstetrical condition.

Commercial interest in the diagnostic capabilities of EVs has been steadily growing.

Companies such as Exosome Diagnostics, miR Scientific, and Ymir Genomics all offer EV-based tests, with Exosome Dx's commercial prospects potentially benefiting from a collaboration between its parent company Bio-Techne and Thermo Fisher Scientific.

The field is also benefiting from a stream of new entrants. Earlier this year, for instance, Sydney-based BCAL Diagnostics announced plans to conduct studies in support of its own EV-based breast cancer diagnostic assay, while Mercy BioAnalytics began development on an EV-based ovarian cancer test.

"I think you'll see more and more EV companies coming along," Tseng said. "To us, this is a very exciting time."