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Sweden's Saga Diagnostics Eyes Clinical Oncology Space With Ultrasensitive Mutation Detection Method

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NEW YORK (GenomeWeb) – Fresh off a $4.2 million funding round, Swedish startup Saga Diagnostics is positioning its mutation-detection technology for use in a variety of cancer-related research and eventual clinical applications, including companion diagnostics, identifying disease relapse, and monitoring cancer therapy response.

The Lund-based firm claims that its technology, enabled by digital PCR (dPCR) and the company's proprietary chemistry, can detect circulating tumor DNA (ctDNA) with a mutational allele frequency (MAF) of 0.001 percent.

Saga CEO Lao Saal and Chief Technology Officer Anthony George founded Saga Diagnostics as a spinout of Lund University in 2016, moving the company into its own lab earlier this month.

Saal said that Saga currently offers two different technologies that researchers can use for genetic and circulating tumor DNA analysis. The firm's IBSAFE platform uses a proprietary chemistry method with dPCR to measure point mutations and indels. According to Saal, researchers can run IBSAFE on any type of input material, including liquid biopsy plasma samples, serum, and cerebrospinal fluid, as well as on conventional tissue samples. Developed using Bio-Rad's QX droplet digital PCR systems, IBSAFE is currently sold as a research-use-only (RUO) kit and as a testing service by Saga.

"In principle, IBSAFE should work on any compartmentalized dPCR system, and we have confirmed this with Stilla's [crystal digital PCR platform] and are in discussions to confirm this on other platforms too," Saal explained.

Saga has also developed a technology called KROMA, which is a hybrid method that incorporates next-generation sequencing (NGS) and dPCR for detecting minimal residual disease, occult metastasis, and to monitor therapy response. Developed using equipment from both Illumina and Bio-Rad, KROMA measures chromosomal rearrangements as quantitative liquid biopsy molecular markers for a patient's tumor mutational burden. Saal noted that KROMA "in principle" should work with other sequencing and dPCR systems.

According to Saal, researchers interested in using IBSAFE can purchase a kit and perform the analysis in their own labs if they have the requisite equipment. Customers can also send a sample in a collection tube to Saga's lab for analysis, which Saal noted is in the process of obtaining International Organization for Standardization accreditation. Researchers can also send in samples that have been processed in their own lab, such as extracted nucleic acid samples.

While IBSAFE requires only dPCR analysis, KROMA requires an additional low-pass whole-genome sequencing and proprietary bioinformatic analysis step in order to count the chromosomal rearrangements and rebuild the exact sequences at the breakpoints.

"Based on this rearrangement 'fingerprint,' our bioinformatics pipeline selects rearrangements and personalized assays are automatically designed for synthesis," Saal said "The panel of fingerprint assays can then be utilized to monitor that patient uniquely for the entirety of their follow up." 

While IBSAFE produces results in about a day, KROMA first requires about a month from initial tumor sampling to develop customized assays. After the WGS step, however, liquid biopsy analysis using the customized assays has a turnaround time of two to three days, Saal claimed.

"If you don't put in enough genomic equivalence in, then what becomes a limiting factor is the sample, not the technology," Saal said. "If you only put in 10 genome copies, and only one is mutated, then that's a 10 percent match, but to get .001 percent, you need to put in at least 100,000 copies. While our blood test works on any blood sample volume, you can only reach 0.001 percent when you have enough of a sample with one copy."

According to Saal, the firm's technology therefore typically uses between 10 and 50 ml of a patient's blood to reach a limit of detection (LOD) of about 0.001 percent.

Kenneth Cole, group leader at the National Institute of Standards and Technology's (NIST) bioassay methods, biosystems, and biomaterials division, is interested in the quality and consistency of the data from the measurements that help detect one variant in a background of 100,000 wildtype molecules, which would allow the use of these ultrasensitive measurements in the clinical space for cancer-based applications.  

In general, Cole said that clinical trials need to be done to prove the clinical utility of a test using ctDNA with a MAF as low as Saga suggests with its assays, which he noted is an order of magnitude lower than possible with currently available technology. Cole highlighted that regulatory agencies like the US Food and Drug Administration have yet to note the clinical significance of such a low limit of detection, and that researchers will need to draw the line in future clinical studies.

Cole also believes that researchers would typically need to use a large volume of a patient's plasma to detect ctDNA at such a low level of variant DNA sequences, noting that there is "a wide range of concentrations of cell-free DNA present in patient samples."

While he acknowledged that researchers could potentially detect traces of cancer in the types of volumes being targeted by Saga depending on ctDNA levels, Cole emphasized that there is a high variance in ctDNA concentrations that occurs in patients' samples based on their type of and stage of cancer.

In a 2015 study published in EMBO Molecular Medicine, Saal and his team used KROMA to retrospectively identify ctDNA in a group of 20 patients diagnosed with primary breast cancer. The goal of the study was to provide proof of principle that the assay could identify recurrence before standard methods.

Extracting ctDNA from 93 plasma samples, they quantified the number of fragments of each tumor-specific chromosomal arrangement in the ctDNA by dPCR. The team was able to detect eventual cases of breast cancer recurrence with 93 percent sensitivity and 100 percent specificity.

Saga is also developing an unnamed third platform that is a pure NGS approach based on gene panels, which Saal said is similar to technology offered by other sequencing companies. He noted that the firm will use the platform to minimize the false positive noise in a cancer sample.

"What will be different [about the third platform] is [that] the library preparation and the bioinformatics …  [will be] augmented by machine learning to improve noise reduction and error correction," Saal explained.

Validation study

In June 2018, Saga signed a customer agreement with the Center for Molecular Diagnostics (CMD) at the Skåne University Hospital system in Scania, Sweden. The CMD, which is part of Clinical Genomics Lund, is now validating Saga's IBSAFE technology for clinical use to aid treatment decisions for patients with NSCLC and AML.

Anders Edsjö, a senior pathology consultant at Region Skåne's Clinical Genetics and Pathology lab, noted that the validation study is still ongoing in both diseases. He explained that researchers have performed labwork and technical troubleshooting in the molecular pathology unit and CMD, which are both in the same department at Clinical Genomics Lund.

Edsjö said that the AML project aims to compare IBSAFE to an unnamed Swedish clinical standard assay performed at the Sahlgrenska University Hospital. While Edsjö believes the results are promising, he noted that the researchers are still finalizing the analysis.

Within the NSCLC project, Edsjö said the group has divided the work according to targeted clinical questions. However, he noted that Saga's assay for detecting EGFR variants in plasma to identify resistance to first- and second-generation tyrosine kinase inhibitor treatment is already in clinical use. Edsjö's team is currently following up with assays for "sensitizing variants and additional resistance mutations to address resistance to third generation TKI and anti-ALK-treatment resistance in NSCLC."

Edsjö mentioned that the group's major challenges have mainly been pre-analytical, adapting DNA-preparation protocols to avoid issues in the downstream PCR step. While the team does not have suggestions for improving Saga's assay, Edsjö noted that an assay for "ALK and ROS1 [biomarkers] would be very valuable" to use plasma testing to identify patients with NSCLC.

"We hope to continuously expand the list of assays, [such as] BRAF for melanoma, PIK3CA for breast cancer, et cetera," Edsjö said. "This requires a close collaboration and a high degree of methodological skills on all parts."

Saal noted that researchers using standard qPCR, dPCR, and NGS have struggled with false positive data in the 0.001 percent to 0.1 percent range because of nucleotide incorporation errors by the polymerase enzyme. He believes that the issue even happens with high-fidelity enzymes, as the low rate of error causes false-positive variants at most positions in the genome. Saal emphasized that IBSAFE's use of dPCR compartments with the firm's proprietary chemistry reduces the consequence of polymerase errors, claiming that it "effectively reduces the noise to near zero."

"For many applications, a digital PCR approach may be better [than qPCR and NGS] when you have companion diagnostic drugs or mutations that are easily understood, and you want results that don't require you to batch several samples together per run," he said.

According to Saal, Saga has signed commercial agreements with several undisclosed pharmaceutical partners and biomedical laboratories. However, Saal declined to disclose the price of the RUO kits because several different factors are involved that will affect the overall cost of the test for researchers.

Saal noted that Saga filed the first patent for its liquid biopsy detection tool in 2015 with "14 different jurisdictions, including the US, Canada, China and other major markets," and anticipates CE-IVD approval for its IBSAFE technology by the end of the year. He also emphasized that the patents related to KROMA and IBSAFE are all wholly owned by Saga.

In addition to last week's $4.2 million venture financing round, Saga Diagnostics has raised about $2 million through academic grants and funding rounds in the past few years, including a $497,000 grant from Sweden's SWElife Innovation program in November 2018 and a $1.3 million March 2018 seed funding round.

Saga will use the recently raised funds to further commercialize its portfolio of kits and services and perform prospective clinical studies. While Saga plans to receive regulatory approval for its KROMA and IBSAFE RUO kits for customers, it will also use the funding to continue developing the third "pure" NGS platform. Saal also noted that his team is in the process of rebranding IBSAFE and KROMA.

NIST's Cole said that there are significant challenges to provide reliable measurements at high levels of sensitivity given the complexities and differences in patient samples. Until Saga's technology has been proven further in peer-reviewed studies for clinical use, he remains skeptical about its use in clinical applications such as early detection, monitoring cancer relapse, and response to drug therapy.

"When you claim to have that level of sensitivity, you need to have a lot of high-quality data from patient samples to back it up," Cole said. "Right now, researchers are pushing the limit of detection for ctDNA, but we don't know what the clinically significant level is yet, and thus it needs to be proven by clinical trials in the future."

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