Circulogene Broadens Push Into Companion Diagnostics with NTRK Fusion Assay

NEW YORK (GenomeWeb) – With the upcoming launch of its neurotrophic receptor tyrosine kinase (NTRK) gene fusion assay, liquid biopsy firm Circulogene is continuing to explore the use of reverse-transcriptase PCR to identify drug targets in cell-free enriched RNA.

Its new NTRK1/2/3 Fusion Test identifies up to 20 clinically relevant fusions between NTRK1/2/3 and other genes, which the firm said is important for cancers with a less than one percent NTRK-positive detection rate.  

Instead of drawing blood using fingerstick sample prep technology, Circulogene has now started using blood tubes for sample collection to improve efficiency and ease of use for it NGS and its new RT-PCR assays.

According to Circulogene CEO Michael Mullen, the Birmingham, Alabama-based firm began developing the NTRK assay in early 2018. The  company aims to eventually launch the service alongside US Food and Drug approval of larotrectinib for cancer therapy.

Circulogene also markets multiple next-generation sequencing-based tests, including one that tracks microsatellite instability (MSI) for immunotherapy, as well as DNA, RNA, somatic, and germline mutations. The tests, which the firm said can produce results in five to seven days, allow clinicians to identify patients eligible for treatment with drugs for conditions such as lung, pancreatic, prostate, breast, and colon cancers.

"Our strategy is to validate precision medicine markers as the oncology drugs clear the market and validate the clinical utility of those in the clinical practice," Mullen explained.

While Circulogene’s NGS platform currently covers several biomarkers, Mullen highlighted that NTRK adds to the firm’s three highly sensitive cell-free RNA PCR-based tests that already exist, including the PD-L1 marker for immunotherapy.

According to Circulogene CSO Chen-Hsiung Yeh, the firm developed and validated the NTRK1/2/3 test on Thermo Fisher Scientific's QuantStudio 6 RT-PCR platform, which runs on a 384-well plate. The test uses 4 milliliters of a patient's blood to detect gene fusions.

Physicians start by selecting an individual test or panel test for actionable information for treatment decisions.  After collecting a patient's blood in a blood tube, oncologists send the sample to Circulogene's CLIA-certified lab in Birmingham.

Circulogene Lab Supervisor Andrew Ford explained that the firm uses a proprietary, extraction-free enrichment process on the blood sample, which "enables us to enrich enough of the circulating cell-free DNA or RNA to really maximize our use of that material in different workflows."  

Ford noted that the firm's lab then performs the RT-PCR assay and can produce results in three days. According to Mullen, the assay detects a total of 20 genetic fusions: 11 NTRK1 fusions, six NTRK2 fusions, and three NTRK3 fusions.

Even though TRK fusion cancers are rarely diagnosed, Circulogene initially plans to use the assay to detect conditions often linked to NTRK fusions, such as infantile fibrosarcoma, thyroid cancer, and high-grade gliomas. Mullen highlighted that doctors also have the option to order the test for additional cancers that could benefit from drug treatment, including pancreatic, non-small cell lung, or colorectal cancers. The firm estimates that about 25 percent of cancer patients tested may then qualify for "some sort of targeted and or immunotherapy."

"Our purpose is to provide ... cancer [patients] and physicians as many treatment options as possible in a timely and cost-effective manner to help patient outcome," Mullen noted. "NTRK is a very important option with its multi-tumor indication, and it fits nicely within our offering based on available precision therapies and National Comprehensive Cancer Network guidelines."  

While Circulogene is unable to patent the NTRK 1/2/3 assay, Mullen noted that the firm has instead filed a patent for its sample prep solution used in the assay. He also said that the firm has performed some research and clinical testing with the University of Alabama at Birmingham since 2016.

According to Mullen, Circulogene will initially offer the NTRK fusion assay as a laboratory developed test for research use only. The firm is currently performing clinical utility studies with several undisclosed academic institutions on the assay "for payor assessment." While unable to disclose information about the assay's clinical sensitivity and specificity, Ford said that Circulogene hopes to publish the clinical utility study by the third quarter of this year.  

Mullen also noted that Circulogene plans to eventually work with a pharmaceutical company to develop a 510(k) FDA-approved companion diagnostic assay.

Despite initially using fingerstick-based sample-prep methods for its assays, Circulogene now collects patient blood samples with venipuncture because the firm saw that the method was much "easier, more convenient, and more popular in the oncology practice," Yeh said. "With fingerstick, [however], you have to keep squeezing blood out and sometimes blood [that's] been clotted impacts sample quality."

In addition, Yeh highlighted that the 4-ml blood sample allows clinicians to save more blood for future genetic tests that detect additional mutations.  

As it prepares to commercially launch the NTRK assay, Circulogene will enter a crowded space for precision medicine-based cancer diagnostics using cell-free nucleic acid assays.

Foundation Medicine received Breakthrough Device designation from the US FDA last April for its liquid biopsy assay, an expanded version of its existing FoundationACT assay. The NGS assay will detect substitutions, indels, copy number alterations, and select gene rearrangements in more than 70 genes, as well as MSI and blood tumor mutational burden in circulating cell-free DNA.

Guardant Health offers its liquid biopsy-based Guardant360 assay, which analyzes about 54 cancer-related genes in a patient’s bloodstream. Since going public last year, the firm has published promising results on the assay's use for detecting mutations in NSCLC patients, reflecting potential real-world applications.   

Mullen believes that Circulogene's NTRK fusion test will stand out from competitors' assays because it will have extremely high sensitivity, with "the fastest turnaround time available, regardless of tumor diagnosis." The firm believes that it is the only provider that can deliver a complete set of results within a week based on NCCN guidelines and available therapies using a single 4-ml tube of blood.

Mullen noted that Circulogene expects to sell the test for about $200 per sample, but he argued that payor reimbursement could "range anywhere from 0 to $200."

While Larotrectinib has shown unique responses in different cancer types,  Mullen explained that the market lacks a highly sensitive and well-known NTRK assay. By combining PCR-based testing with its proprietary liquid biopsy sample-prep technology, Circulogene believes its NTRK test can be the most sensitive assay available.

Hematologist Stephen Eck, who serves on Circulogene's board of directors, noted that the firm will need to determine the economic utility of the NTRK assay before implementing them on a larger scale.

"There are certain genes that you're looking for that don't really have a lot of medical economic utility," Eck explained. "So [it] will take time to understand relationship between what you measure, what the medical intervention was, and what the outcome was."  

In general, Eck believes that RT-PCR is being used more extensively for multiplex cell-free nucleic acid tests. He sees Circulogene's blood-tube approach as cost-effective for clinicians aiming to "look for several mutations simultaneously rather than one at a time."

As it plans to launch the NTRK assay, Circulogene said last week that it has partnered with its Auckland, New Zealand-based subsidiary to expand its footprint into the Pacific-Asian region.  

"We're trying to be efficient by having patients tested, previously forming a subsidiary, and now actively collecting samples from Malaysia," Mullen explained. He said the group had successfully identified a rare cancer in one patient and is now working with the clinician on the best avenue for the patient's treatment.

In addition to detecting and monitoring cancer mutations, Circulogene is also exploring options for its technology in other fields, including non-invasive prenatal testing, organ transplantation, infectious disease, and biodefense. Mullen noted that the firm is now involved with several military projects as well as working with US Department of Defense, but he declined to provide additional details.

Eck also added that while “cancer's a great place to start, as the molecular biology of the [disease] is slowly being understood at a detailed level … we will see the same thing happening with other disorders, where their underlying etiologies will be quite different."

For example, once researchers distinguish the genetic discrepancies behind common mental health issues like schizophrenia, depression, autism, and other neuroscience disorders, Eck believes that they could eventually use a panel of genetic tests developed by companies like Circulogene to tailor treatment to a patient's specific health requirements.