NEW YORK – A group of researchers at the University of Virginia has evaluated Ceres Nanosciences' magnetic capture technology for the isolation of circulating tumor DNA (ctDNA) from other genomic material in blood samples.
While Manassas, Virginia-based Ceres is currently focused on developing sample prep products to improve sensitivities of COVID-19 diagnostic assays, the firm said that it is also adapting its Nanotrap platform to capture previously undetectable levels of ctDNA from blood from non-small cell lung cancer (NSCLC) patients.
Nanotraps are hydrogel nanoparticles functionalized with internal affinity baits to enrich target analytes for downstream analysis. Eli Williams, UVA associate professor of pathology and senior director of the university's Clinical Genomics Lab, explained that the technology selects particles based on size and affinity properties for nucleic acids. Nanoparticles bind to targets and concentrate them inside their shells to protect against degradation, which improves the sensitivity of downstream detection methods.
"Essentially, the hydrogel can be modified, depending on the particular biomolecule you're trying to enrich for," Williams explained. "We've modified it to increase for affinity of nucleic acids, and the shell can exclude larger genomic DNA or other contaminated biomolecules."
Williams' team began exploring liquid biopsy extraction methods with Ceres in 2019. The collaborators, which also involve researchers at George Mason University (where Ceres was founded), received a one-year $600,000 grant from Virginia Catalyst in April of that year to test and develop Nanotrap for the liquid biopsy space.
In a proof-of-concept study presented at the 2020 virtual Association for Molecular Pathology annual meeting last month, Williams' team compared the recovery of ctDNA using the modified Ceres Nanotrap tool and the Cobas cfDNA Sample Preparation Kit from Roche Diagnostics. The group focused on EGFR-specific mutations due to their clinical relevance in NSCLC.
In the study, the researchers initially created contrived liquid biopsy specimens by spiking fragmented EGFR wild-type and mutant DNA sequences and Horizon standard ctDNA into pooled donor plasma samples at varying concentrations. They then extracted the samples using both technologies, followed by several downstream mutation-detection methods to test Nanotrap's isolation abilities, including Roche's Cobas 4800 PCR assay.
The researchers further compared both extraction methods using two cohorts of NSCLC patients with known mutational profiles. Collecting 10-ml blood and matched tumor tissue samples from 24 patients from UVA's Biorepository and Tissue Research Facility, they then extracted ctDNA from plasma containing ctDNA using the methods.
"We've actually compared Nanotrap to other technology on the market, but we showed data on Roche's because our initial studies were using Roche's EGFR assay," Williams said. "We wanted to show that the Roche pipeline was optimized as a benchmark for our Ceres approach."
The group then extracted ctDNA from a cohort of eight NSCLC patients and tested a panel of 28 genes — including EGFR, BRAF, KRAS, and other common mutations — using ArcherDx's next-generation sequencing (NGS) panel.
The researchers found that the modified hydrogel approach had an overall improved average ctDNA recovery (79 to 84 percent) than the Cobas cfDNA kit (48 to 63 percent) at different levels of spiked DNA from plasma samples.
Williams highlighted that the Nanotraps prevented genomic DNA contamination for up to 24 hours, which he believes is crucial for clinical workflows. The group also successfully detected a variety of clinically useful EGFR mutations using Nanotrap down to biologically relevant concentrations.
While Williams' team is still optimizing Nanotraps for ctDNA extraction purposes, he believes the tool is compatible for downstream diagnostic tests. Williams said that the method requires about four hours to extract ctDNA from blood samples, and that it is effective at helping prepare nucleic acids for downstream detection of EGFR molecular profiles in NSCLC patients.
The researchers are now aiming to increase the cancer patient cohort size and include additional data from NGS results to publish the full results of the study next spring. Williams noted that the group is partnering with ArcherDx and its 28-gene NGS panel as part of an eventual multigene liquid biopsy workflow to detect cancers, including colorectal cancer.
"Now we're starting to expand to next-generation sequencing-based platforms to broaden both diagnostic yield and clinical utility of a downstream liquid biopsy assay using Nanotrap," Williams said. "We initially used Roche's EGFR panel as our downstream comparison assay, but we're trying to improve the clinical utility of the test for our patient population ... [by eventually] having a multi-gene panel."
While Ceres has focused most of its efforts this year incorporating Nanotrap reagents into a number of labs' SARS-CoV-2 tests to address the COVID-19 pandemic, CEO Ross Dunlap said in an email that the firm has kept "key resources dedicated" to develop the Nanotrap liquid biopsy product.
Besides Roche, other major competing ctDNA extraction methods include Qiagen's QIAamp circulating nucleic acid kits and Norgen Biotek's Plasma/Serum cell-free circulating DNA Purification midi kit. Promega's Maxwell RSC ccfDNA Plasma Kit and Zymo Research Quick ccfDNA kit also allow users to extract cell-free circulating DNA from patient plasma samples.
In a 2018 comparison study, researchers found that, in general, spin column-based extraction kits performed better than more recently introduced magnetic bead-based kits in their ability to extract circulating cell-free DNA from patient blood samples.
Irvine, California-based startup nRichDx is also targeting the liquid biopsy space with its magnetic sample prep Revolution System, which collects a variety of genetic material such as ctDNA.
Dunlap noted that Ceres has a "robust" patent portfolio and trade secrets that cover a wide range of applications for the Nanotrap technology, including nucleic acid capture.
"Our workflow has fewer steps and hours at the bench and delivers more ctDNA with less gDNA contamination [than existing technology]," Dunlap said. "We will continue to move this product development effort forward with an aggressive timeline in 2021."