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Agena Bioscience Shows Off New Liquid Biopsy QC Assay at AMP; Expects January Launch

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NEW YORK – Agena Bioscience is preparing to commercially launch a pre-analytical quality control assay that it expects to increase the efficiency of liquid biopsy testing.

In a corporate workshop presentation at the 2020 virtual Association for Molecular Pathology annual meeting this week, an early-access user provided a sneak peek of validation data for the assay, called Liquid IQ, and how it might be used in combination with the company's UltraSeek circulating tumor DNA (ctDNA)-based lung cancer mutation panel.

The firm also discussed the results of recent studies favorably comparing the research-use-only UltraSeek panel to more established liquid biopsy assays to detect drug-resistant mutations in non-small cell lung cancer (NSCLC).

Agena's Liquid IQ assay determines the quality and quantity of cell-free DNA (cfDNA) in a patient's blood sample. The assay can produce results in eight hours using 1.5 μl of DNA from 50 μl to 100 μl of ctDNA extracted from the sample.

During the workshop, Ed Schuuring, professor of molecular oncological pathology at the University Medical Center Groningen in the Netherlands, explained that the assay's report provides easy identification of total amplifiable copies, white blood cell contamination, presence of long DNA fragments, and sample matches or mismatches. The report also lists samples that may have failed quality control due to poor- or low-quality DNA.

In a study provisionally accepted by the journal Cancers, Schuuring and his colleagues used Liquid IQ to assess and quantify circulating cfDNA used in Agena's UltraSeek Lung Panel. The team selected 91 blood samples from 48 NSCLC patients representing random disease stages, timepoints, and therapies. The group then compared circulating cfDNA yield using Liquid IQ to results obtained with Thermo Fisher Scientific's Qubit fluorometer assay kit. 

Schuuring presented data from one sample assessed with Liquid IQ that included the amount of SNP calls, amplifiable copies, and most importantly how many microliters of DNA a user should have for an input volume of 10 ng of amplifiable cfDNA for a downstream assay. He also highlighted that there was a high correlation between the test and Qubit.

Schuuring believes that Liquid IQ may help with quality assessment and quantification of circulating cfDNA, which is typically "quite laborious to perform."

"Because PCR reactions may be biased to larger fragments, the [test] could determine the presence of high molecular weight fragments in the sample," Schuuring explained. "The test also includes SNP markers to control for mix-ups" that can complicate results.

In a follow-up email, Darryl Irwin, VP of scientific affairs at Agena, explained that the firm provided the Liquid IQ assay to "key collaborators" in 2019 as an early-access product. Agena has since optimized the assay to increase sensitivity toward white blood cell and long-fragment DNA contamination.

Irwin said that Agena expects to launch the optimized product as the iPlex Pro Liquid IQ panel in January, with the goal of enabling users to identify multiple issues before "starting an expensive liquid biopsy workflow."

NSCLC comparison studies

Schuuring also recapped his group's recent comparisons of different ctDNA-based methods to identify NSCLC patients who may develop resistances to EGFR-tyrosine kinase inhibitor therapies such as osimertinib (AstraZeneca's Tagrisso).

Agena's UltraSeek Lung assay, which runs on the firm's MassArray endpoint PCR- and mass spectrometry-based platform, identifies somatic mutations in ctDNA and circulating tumor cells (CTCs) in plasma and solid tumor tissue. The assay can use DNA extracted from 10 ml of a sample and can detect mutations at levels as low as 0.1 percent mutant allele frequency.

As part of one study, published in June in Cancers, Schuuring's team performed a multicenter evaluation of UltraSeek with a broad range of next-generation sequencing (NGS) and PCR-based detection methods. NGS panels included Roche Diagnostics' Avenio ctDNA Targeted kit, Qiagen's QIAact Lung UMI Panel, and an Agilent SureSelectXT custom kit. Bio-Rad Droplet Digital PCR (ddPCR) assays included one that was ISO15189-certified (ISO) and another test that was for research use only. Using SeraSeq reference material, the team analyzed 25 variants, including 12 single nucleotide variants, five deletions, three copy number alterations, and three fusions, at a variant allele frequency (VAF) of 1 percent.

Schuuring said in an email that his team saw that UltraSeek demonstrated the lowest variability when compared to the other methods, with a variability of 19 percent at 15 ng of DNA. The ISO and RUO ddPCR assays had comparable variability of variant calls using 8 ng of input (24 and 30 percent respectively). Meanwhile, Roche's Avenio assay and Agilent's SureSelect had about a 22 percent and 26 percent variability respectively at an input of 20 ng. However, Qiagen's QIAact had the highest variability at 48 percent at an input of 20 ng.

"The study found that the MassArray was the most precise technology assessed and therefore most ideally suited to quantifying mutation frequencies in liquid biopsy samples," Irwin added. He pointed out that the measured variability was equal to or slightly higher than the expected variability, except for UltraSeek, which the researchers found to be "more precise than expected."

In a second study, published last month in Cancers, Schuuring's team compared UltraSeek with Roche's Cobas EGFR Mutation Test v2 — which recently received approval from the US Food and Drug Administration as a companion diagnostic for NSCLC therapies — to detect EGFR mutations in liquid biopsies, in tandem with preanalytical assessment using Liquid IQ. The group used UltraSeek Lung to test 137 cancer patient plasma samples previously analyzed with the Cobas EGFR assay, using Liquid IQ to determine circulating cfDNA input for all samples.

While overall concordance between the two tests was 86 percent, the Cobas test detected more EGFR exon19 deletions and L858R mutations, while UltraSeek detected more T790 mutations. By using an input of 10 or more ng of circulating cfDNA, the team found that UltraSeek showed 100 percent concordance with the Roche test.

Schuuring noted that UltraSeek also detected additional mutations not covered by the Cobas panel, including treatment resistance mutations such as KRAS mutations and BRAF.

The group believes that UltraSeek could be eventually be used to identify resistance mechanisms in NSCLC patients during EGFR-TKI treatment, as well as to make an early diagnosis when oncologists cannot access tumor tissue.

Schuuring acknowledged that UltraSeek is relatively expensive compared to other liquid biopsy tests available for initial diagnosis of NSCLC. However, he believes that the test could serve as a parallel service to tissue testing to rapidly identify the most common and clinically relevant mutations in a NSCLC patient's sample, quickly guiding clinical treatment decision making.

"When it comes to performing routine ctDNA mutation analysis, UltraSeek offers the required sensitivity across multiple genes at a low cost and a fast turnaround time," Schuuring said. "This will be especially important when it comes to treatment and resistance monitoring, where patients are tracked at regular intervals for the emergence of resistance mutations."

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