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U of Kansas Researchers, BioFluidica Develop Microfluidic-Based Diagnostic for Cancer

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NEW YORK (360Dx) – Researchers at the University of Kansas have developed a microfluidic technology to detect and isolate circulating plasma cells (CPCs) in patients diagnosed with clonal plasma cell disorders (PCD) including multiple myeloma.

BioFluidica, a San Diego, California-based startup, is using the technology for research purposes, but plans to submit an application to the US Food and Drug Administration for marketing clearance.

While the technology can be used for a variety of cancer types, for now the emphasis will be on multiple myeloma, according to the researchers. Often undiagnosed until later stages, multiple myeloma causes white blood cells to produce abnormal antibodies. The plasma cells can also form a mass in the bone marrow or soft tissue. In advanced stages, the condition may cause bone pain, bleeding, frequent infections, and anemia.

Researchers typically detect multiple myeloma based on blood or urine tests that find abnormal antibodies. In addition, they can search for cancerous plasma cells in the patient's bones using invasive bone marrow aspiration and biopsies. However, each tissue biopsy can cost up to $5,000 and can only be performed every six months due to the pain it causes the patient. In addition, many cancers like multiple myeloma can metastasize in the period between biopsies and potentially kill patients between each diagnostic test.

In a study published in Integrative Biology last week, the researchers evaluated the ability of a sinusoidal microfluidic device to affinity-select CPCs for different PCDs, as well as to release CPCs and perform cytogenetic and molecular analysis on the enriched cells. Stephen Soper, chemistry and mechanical engineering professor at the University of Kansas, tested blood samples from 47 patients with different plasma cell disorders — with monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma — and five healthy donor samples that acted as negative controls.

The team loaded the whole-blood sample on to the chip and then processed it by flowing the chip at the appropriate flow velocity to "maximize recovery of the rare cancer-associated cells." In order to detect multiple myeloma, they enriched the circulating plasma cells using CD138, which has previously been found to be expressed higher in diseased CPCs.

Soper noted that different antibodies can be used with the chip to target different types of circulating tumor cells. Once the chip enriches or captures the cells, researchers can wash and release the cells from the channels' surface.

In the study, the team analyzed the different blood samples using CD138 monoclonal antibodies attached to the surface of the microfluidic channels. Soper explained that the microfluidic chip, which is about the size of a credit card, uses about one to two milliliters of the patient's blood. The chip requires minimal sample prep, and researchers can expect results in less than two hours.

"We can decorate the chamber with antibodies specific for the focused disease," Soper explained. As the blood moves through the microfluidic channels, the targeted plasma cells bind to the antibodies, while the rest of the unwanted cells float by.

Overall, Soper and his team identified CPCs in 78 patients with MGUS and all patients with multiple myeloma. In order to definitively state that they were cancer cells, the team stained the recovered cells with unique markers targeting a specific protein found in the target cancer cell.

According to Soper, the research group discovered that the test's sensitivity was 100 percent for patients with symptomatic and smoldering multiple myeloma, and 78 percent for individuals with MGUS. In addition, they saw that the clinical specificity was 100 percent in all patient cases.

The researchers also found that the microfluidic chip had a CD138-expression CPC recovery rate of 69 percent and sample purity of 68 percent. The authors noted that the metrics are "crucial because the test provides the ability to search for rare target cells ... and perform molecular analysis on the enriched cells without requiring single-cell picking."

Performing KRAS mutational analysis in three clinical samples isolated from samples, the team found point mutations including different mutations from sub-clones derived from the same patient. The team then performed fluorescent in situ hybridization and revealed the presence of chromosome 13 deletions in CPCs linked to bone marrow results.

In terms of clinical utility, Soper believes that researchers could use the test for most solid tumor cells, as well, if placed in solution. In addition, his team is also working on improving the technology in order to extract neonatal cells from maternal whole-blood samples.

While developing the microfluidic chip for research and commercial use, Soper noted that his team has encountered two major obstacles. While the researchers were able to examine a small number of patients in the study, Soper acknowledged that scaling up the test for massive chip production was a challenge for his team. In addition, the team initially struggled to develop a recovery technology for the targeted cells bounded by the antibodies.

Additionally, Peter Voorhees, study co-author and member of the Levine Cancer Institute in Charlotte, North Carolina, explained that once the team captured CPCs, actual cell counting was manually performed by one of Soper's investigators. Voorhees believes that the group will eventually need to automate the enumeration process to produce results more quickly. In addition, he noted that the microfluidic chip's rate of cell capture and purity levels could improve by altering the width of the channels or tethering additional antigens that also bind to cells of interest.

Voorhees, however, highlighted that the blood test will allow researchers to simultaneously assay blood samples from multiple spots where multiple myeloma may develop without having to subject the patient to repeated painful biopsies.

"Ideally, we'd move away from subjecting patients to serial bone marrow biopsies completely," Voorhees explained. "[But] we will need to prove what we're seeing in peripheral blood matches what we see in bone marrow samples."

In order to further develop a diagnostic tool that uses the microfluidic chip technology, Soper cofounded Biofluidica with Rolf Muller, Biofluidica CEO, in 2016. The firm has licensed Soper's CTC, cfDNA, and exosome technology, in addition to sample-to-answer, on-chip analysis technology developed by Louisiana State University, Cornell, and the University of North Carolina.

Muller explained that by using injection molding, the firm can manufacture them for $2 per plastic chip. The chips are programmable toward any kind of clinically or diagnostically relevant cells, and Muller said that the firm has clinically validated the technology for a variety of cancers: lung cancer, breast cancer, prostate cancer, pancreatic cancer, colorectal cancer, bladder cancer, leiomyosarcoma, cholangiocarcinoma, and appendiceal mucinous neoplasm, as well as acute myeloid leukemia (AML) and multiple myeloma.

"We can be tremendously cheaper, faster, and cause a better outcome than standard tissue biopsies, which cost $5,000 per test for each patient," Muller said. "There are inherent risks associated with these tests, as you're putting large needles in people to extract bone marrow."

Soper originally filed a patent for the microfluidic chip technology in 2009. Since then, the teams at KU and Biofluidica have been developing the blood-based test for a variety of cancer diseases, including multiple myeloma. In an email, he explained that he has "filed for and has been granted several new patents that further elaborate on the microfluidic chip technology." He highlighted one that "is for using a unique marker for different types of cancer cells that have high aggressive natures to facilitate the onset of metastatic disease."

Biofluidica initially received extensive Small Business Innovation Research funding from the National Institutes of Health for AML, multiple myeloma, and ovarian cancer, and now plans to eventually apply for 510(k) approval from the FDA for the microfluidic chip. Muller, however, declined to comment on the test's exact commercialization timeline. Since 2016, the firm has raised $7 million through Series A and B rounds of funding involving and angel investors, he said.

While Biofluidica's test is currently for research use only, Muller explained that the firm has partnered with three academic institutions to screen patients with different forms of plasma cell disorders. Developing the plastic chips in house, Biofluidica then delivers them to its CLIA-certified/CAP accredited labs, core facilities in San Diego, Lawrence, Kansas, and Chapel Hill, North Carolina, where they process and analyze patient samples on the system to detect different types of cancers. In addition, Biofluidica has partnered with Soper's team at the Children's Mercy Hospital in Kansas City to diagnose cases of acute leukemia in pediatric patients.

Biofluidica, however, is not the only player in molecular diagnostic market separating circulating tumor cells in patients using microfluidics. Recent startup Akaduem is developing a sample collection tool using its microbubble technology, which uses low-density, air-filled bubbles coated with specific antibodies to bind to CTCs and float it to the surface. UK-based diagnostics firm Angle's Parsortix uses microfluidics with a disposable cartridge to capture and harvest CTCs from blood based on their less-deformable nature and large size compared to other blood components.

Soper argued that the microfluidic chip his team has developed will stand out from competitors because of its high cell recovery rate and purity, as well as the company's ability to scale and mass produce the chip for clinical applications. In addition, Muller noted that the technology has "no cell loss because of controlled sample handling, no pre-processing, and complete reduction of stress on the rare cells avoiding cell rupture."

Mehmet Toner, a faculty member at Massachusetts General Hospital and a professor of bioengineering at Harvard University School of Medicine, highlighted that because Biofluidica's microchip uses a positive-selection methodology, researchers need to know specific information about the tumor's phenotype and use appropriate antibodies to pull out the targeted cells.

"It's a little less of an issue [for] blood cells, but for solid tumors it's a bit more complicated, because with each cancer, you need to figure out new positive selection strategies," Toner explained. "If you have a lot of non-specific contaminating cells with the small amount of targeted cells, you can get a lot of non-specific false positives, which [researchers] may need to look out for."

Toner also pointed out that researchers need to select a specific disease or type of cancer they're searching for ahead of time, which he believes may potentially limit a specific microchip's flexibility.

At the same time, Toner said that Biofluidica's test, along with microfluidic technology in general, is a "great way of moving things forward to be used as a clinical tool, as opposed to more traditional, centrifugation-associated processes, which require a lot of technical skill in a robust manner." He agreed that the chip has all the elements to be a useful point-of-care, clinical diagnostic based on its precision, robustness and scalability.

In the meantime, Muller said that Biofluidica's next goal is use future partnerships to expand globally to supply patients with access to its liquid biopsy platform. He envisions clinicians using the instruments in large clinical research organization centers around the world, screening for cancers including multiple myeloma.