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Qiagen, Exosome Dx Building Urine-Based Digital PCR Workflow for FGFR Mutations in Bladder Cancer

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NEW YORK – Qiagen and Exosome Diagnostics, a subsidiary of Minneapolis-based Bio-Techne, said this week that they are developing a new workflow that integrates digital PCR and exosome-based liquid biopsy to detect bladder cancer mutations in urine samples.

The firms expect to present the full results of a clinical comparison study using the workflow to detect FGFR mutations in urothelial cancer patients at the American Society of Oncology (ASCO) virtual meeting  in June.

The bladder cancer workflow is an early demonstration of the types of clinical applications Qiagen is pursuing on its recently launched QIAcuity digital PCR (dPCR) research-use-only system. 

During a session at the Virtual Molecular Med Tri-Con Conference on Tuesday, Ronny Kellner, senior scientist of digital PCR assay development at Qiagen, discussed barriers of current tissue-based testing in urothelial cancer and plans for the new workflow.

"Cytoscopy is required on a regular basis for urothelial cancer patients and [is] a painful procedure requiring anesthesia, often resulting in damage to the urethra and bleeding," Kellner said in a follow-up email after the presentation. "Urine-based FGFR testing would remove that adoption barrier and allow frequent retesting …  [and] creating a new market for monitoring tumor progression and resistance over time."

Kellner said that Qiagen's QIAcuity system contains the QIAcuity dPCR instrument, Nanoplates, Evergreen and Probe PCR kits, and software suite for downstream analysis. The system splits a single PCR reaction into thousands of smaller reactions within individual "partitions." During endpoint PCR using assays that integrate fragment probes, only the partitions that contain a target produce a fluorescent signal.

"Using the number of positive and negative partitions, one can calculate the absolute number of molecules in the analyzed volume," Kellner explained in his Tri-Con presentation. "This entails several advantages, including, absolute target quantification, higher tolerance to inhibitors, increased analytical sensitivity ... and higher reproducibility."

Kellner pointed out that partitioning, cycling, and imaging are all fully automated on the system, and each nanoplate contains 24 or 96 larger wells that contain between 8,500 and 26,000 partitions. He said that the platform produces results in less than two hours and that anywhere between 2 and 200,000 copies can be reliably detected and quantified per reaction.

Kellner noted that the proposed workflow will help solve some of the challenges with Qiagen's tissue-based RT-PCR Therascreen FGFR kit, which the firm uses to identify alterations in the FGFR2/3 gene in urothelial cancer patients to guide treatment with Janssen Pharmaceutical's metastatic bladder cancer drug erdafitinib (Balversa). The test covers four point mutations and five fusions in tissue-derived RNA.

"The Therascreen workflow is simple, but it comes with certain disadvantages because it relies on invasive tissue biopsy," Kellner said. "The new workflow will combine exosome-based liquid biopsy and digital PCR"

The new workflow involves collecting up to 20 ml of a patient's urine rather than traditional formalin-fixed paraffin embedded tissue. Exosome Dx's RNA isolation tool is used to extract exosomal RNA, and complementary DNA is synthesized using Qiagen's Therascreen FGFR RT-PCR kit. The sample is then analyzed using the digital PCR Therascreen FGFR assay.

For comparison, the group will analyze urine samples in parallel on Qiagen's Therascreen FGFR RGQ PCR kit using Qiagen's Rotor-Gene Q qPCR instrument to identify the patients with FGFR2/3 mutations. The firms will also compare urine-based results with results from conventional tissue-based biopsy FGFR screens.

Daniel Enderle, scientific director of Exosome Dx, said during the talk that to test the workflow the firms have designed a clinical study that will look at about 50 patients with urothelial cancer. The team will collect matched urine and tissue samples and clinical data from German molecular pathology company Stratifyer's clinical network.

Enderle said that the team will collect between 5 ml and 20 ml of urine sample from patients and split them into two sample cohorts: one tested with the Therascreen RGQ tissue workflow and one with the urine-based exosomal RNA digital PCR workflow.

"We're going to [establish the] concordance [of] tissue versus liquid biopsy, and we also have experimental data on FGFR3 biomarker expression," Enderle said.

While the firms will present the study's full results at ASCO, Enderle provided preliminary data from a test pilot cohort of 11 urothelial cancer patients. He noted that initial research data "showed promising results" using urinary exosome RNA, with 100 percent concordance with patient tissue.

"We detected a [few] more mutations in the exosomal RNA than we did in the tissue, but it will be more interesting to look at that … [in] the larger study," Enderle added. 

Qiagen and Exosome Dx expanded an existing exclusive collaboration and license agreement in September. 

"The fact that urological biomarkers can be contained in exosomes motivated our initial feasibility study to demonstrate whether with Qiagen's … platform combined with new developments in exosomal isolation and RNA purification from Exosome Diagnostics we could detect and match the mutational concordance tissue taken from the same patient," Kellner said in the email. "A urine-based test would have a higher adoption rate and result in increased testing, which might very well lead to earlier detection of malignant lesions and increased study enrolment."

Kellner said that Qiagen intends to explore other minimally invasive approaches to biomarker analysis  using liquid biopsy technology.

While Kellner could not comment on Qiagen's specific plans and cancers it would pursue in the future, he noted that the firm will work closely with its pharmaceutical partners "where there is a need and interest to expand market access."