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Angle Testing Integrated Cell Sorting, Molecular Analysis Tech for Ovarian Cancer Detection

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NEW YORK – Liquid biopsy developer Angle is developing a version of its Hybrid Capture Enrich Amplification and Detection (HyCEAD) Ziplex molecular analysis technology that — in combination with its Parsortix cell-sorting system — the firm believes could provide a complete sample-to-answer solution for cancer diagnosis. 

The company's first application of the combined technology is ovarian cancer detection. After demonstrating the ability to retrospectively discriminate between ovarian cancer patients and healthy subjects in a 54-patient clinical verification study earlier this summer, the UK-based firm is now about to launch a 200-patient study to examine patients with a pelvic mass who are scheduled to undergo surgery.  

The HyCEAD Ziplex technology, originally developed by Toronto-based Xceed Molecular, switched hands to University of Toronto spinout Axela when it bought Xceed in 2010. After collaborating with Axela to develop multiplex molecular analysis technology for use with Parsortix, Angle acquired Axela in 2017 for $4.9 million, obtaining the rights to the platform to expand its own liquid biopsy capabilities. 

According to Angle CEO Paul Smith, the firm has spent the past two years optimizing HyCEAD Ziplex by improving its stability and producing an in vitro diagnostic assay workflow. Angle has also updated the technology so that researchers can now in a cartridge-based format for immediate testing.  

"Within the liquid biopsy space, our focus is to optimize [HyCEAD-Ziplex] for high sensitivity and specificity," Smith said. "We have fully automated the platform in preparation for a Phase I clinical trial, which will be performed with a reference lab partner or one of our own labs."

After running up to 10 ml of blood through the Parsortix cell-capture system, researchers harvest circulating tumor cells and other rare cells into a storage buffer and begin the HyCEAD process, which uses universal primers to bracket sequences to produce copies and allow simultaneous amplification of more than 100 targets in a single PCR reaction. 

"A strength of this technique is that while it involves high multiplexing … the products going into PCR only needs a single PCR condition, and that stays the same regardless of which genes we choose … giving you more flexibility in changing panels," Smith explained.

A fraction of the PCR product then hybridizes to probes directly on the Ziplex instrument, an automated microarray platform for biomarker detection of gene and protein expression. Ziplex uses the firm's proprietary Flow-Thru Chips and chemiluminescent detection to capture targets from the sample, which Smith argues translates to higher sensitivity or speed compared to traditional microarrays. 

"You have a series of spots arrayed on a surface, each one with a complementary oligo target of what you're trying to measure," Smith explained. "What's different [from typical slide-based arrays] is that the substrate consists of densely packed 10-μm pores to greatly increase the surface area for each spot. Passing the sample back and forth through the array provides intimate contact between the target and surface, resulting in efficient and rapid binding." 

Researchers then analyze the images using the embedded Ziplex software to report the intensities of each gene probe and assay control. 

According to Smith, it takes about six hours to produce results, with the Ziplex detection and analysis step taking about two to three hours.  

In June, Angle announced positive results from a study of Parsortix and HyCEAD Ziplex's combined ability to detect ovarian cancer in women. In addition, the company wanted to see if it could broadly deploy the tool and ship samples without losing testing accuracy. 

Partnering with Rich Moore, an obstetrics and gynecology professor at the University of Rochester Wilmot Cancer Center, Angle examined samples from 26 stage III and IV ovarian cancer patients and 28 healthy controls. Using the Parsortix system, the university researchers processed the samples to harvest CTCs and then sent them to Angle's lab in Toronto. From there, the firm used HyCEAD Ziplex to analyze the cells and detect mutations linked to ovarian cancer. 

The researchers successfully identified samples from healthy volunteers and ovarian cancer patients, though the company did not provide the level of accuracy and has not published results of the study. In addition, the researchers found that they were able to process and transport patient blood samples fast enough to not significantly compromise the assay's sensitivity. Angle will start a larger prospective clinical verification study later this month in partnership with the University of Rochester team to further demonstrate the combined tool's potential in ovarian cancer detection.

In the study, the team will collect samples from 200 women presenting with a pelvic mass prior to biopsy surgery and will use a panel of about 100 genes potentially linked to mutations causing ovarian cancer. 

"Our main question to answer is who you can predict will have a benign or malignant mass, which has a major impact on the surgeon the patient is sent to," Smith explained. "The nature of ovarian cancer requires a specialized surgeon, as it can metastasize rapidly if you don't remove it all effectively." 

The team will also see if could the panel can help identify different phenotypes of ovarian cancer. Smith believes the ability to determine ovarian cancer subtypes would potentially be useful in guiding neo-adjuvant therapy, reducing tumor size, and simplifying tumor removal surgery. 

Angle anticipates completing the verification study by early 2020 to support the launch of a clinical assay for ovarian cancer detection.

By combining Parsortix and HyCEAD Ziplex, Angle aims to offer a combined method for cancer cell isolation and downstream analysis that it believes could also be applied to other cancers, such as breast and prostate. The firm hopes to partner with large healthcare companies to develop multiple clinical applications for the workflow. 

Smith argued that Parsortix and HyCEAD Ziplex will stand out from other PCR methods or sequencing because the tool does not require nucleic acid extraction, as well as its high specificity. 

"A challenge with CTCs is that you only get a single cell when you harvest the solution [after Parsortix], with a background of white blood cells that can range from 1,000 to 10,000 cells," Smith said. "This is a very big challenge for sequencing, as the depth of sequencing [needed] to look at gene expression of that one cell in that background … makes it extremely expensive and complex." 

In contrast, Smith said that the ability to offer a cartridge-based format will make the workflow available to low- to moderate-complexity labs, rather than needing to send samples off for sequencing in a costly and specialized facility.   

"[We want] to make this kind of liquid biopsy available for routine monitoring on a cost-effective and practically available basis in addressable markets, such as prostate and breast cancers," Smith noted. "We believe sequencing will be limiting as an alternative to answering those specific questions on a routine basis and therefore think this is an area that we will be able to offer some advantages." 

Angle plans to apply for 510(k) approval from the US Food and Drug Administration for Parsortix for metastatic breast cancer collection by the end of this year. While Angle currently offers HyCEAD Ziplex as a service to collaborators and pharmaceutical clients, Smith believes it could also be incorporated into laboratory-developed tests prior to the company eventually submitting it for regulatory approval for specific clinical assays. 

"The reason we're developing a cartridge-based platform is so that we could go through regulatory clearance on the technology and provide it as a product or as an instrument that sits in a lab and could locally run a cancer circulating tumor DNA mutation panel that were accessed locally," Smith explained, although he did not provide a timeline for potential regulatory submission.

Smith also declined to provide a specific price range for assays based on HyCEAD-Ziplex, but said he expects it would compare to the cost of other PCR assays. Smith believes that Angle's combined platform can act as a middle ground between PCR and sequencing for answering specific clinical questions.