February 17, 2021 | An innovative interrogation technique developed at Virginia Commonwealth University (VCU) is targeting genetic mutations that are neither very short nor very long and therefore get missed by existing diagnostic approaches. Most rearrangement mutations implicated in cancer and neurological diseases fall between what can be detected by DNA sequence reads and optical microscopy methods such as fluorescence in situ hybridization at the “ends of this barbell,” says Jason Reed, Ph.D., a member of the cancer biology research program at the VCU Massey Cancer Center and associate professor in the department of physics at the VCU College of Humanities and Sciences.
The new technique combines digital polymerase chain reaction (PCR) and high-speed atomic force microscopy (HSAFM) to create an image at a nanoscale resolution that allows users to measure differences in the lengths of genes (aka polymorphisms), explains Reed, who is leading development efforts. It is designed to detect any kind of rearrangement that changes the size of a specific segment of DNA, with an addressable range between five and 5,000 base pairs.
Digital PCR is great at “providing quantitative information about the abundance of minority species of DNA molecules,” Reed says, “but it doesn’t tell you the size of the mutations you are looking for.” It inherently generates many small PCR reactions that leaves little material to further interrogate for size changes.
The problem is solved by coupling digital PCR with single-molecule imaging, allowed by HSAFM, which “can size the DNA molecules in all of these little micro-reactions very accurately,” he says. The combo effectively expands the applicability of digital PCR to a whole new class of mutations, including those responsible for inherited conditions such as Huntington’s disease, Fragile X syndrome, and neurological ataxias.
The nanomapping technology was previously described in a 2017 study published in Nature Communications (DOI: 10.1038/s41467-017-01891-9). The research team showed how HSAFM, used in combination with a CRISPR-based chemical barcoding technique, could map DNA nearly as accurately as DNA sequencing while processing large sections of the genome at a much faster rate.
In a pilot trial recently published in ACS Nano (DOI: 10.1021/acsnano.0c05897), the research team proved out the diagnostic concept in terms of the required workflow, results produced, and compatibility with real medical samples from patients with acute myeloid leukemia. The combined digital PCR and HSAFM methodology matched the performance of the LeukoStrat CDx FLT3 Mutation Assay, the current gold standard test using capillary electrophoresis, in measuring the size of mutations.
Unlike the current test, Reed notes, the new approach also reports the variant allele fraction measuring the relative abundance of a mutation regardless of its prevalence across the cells being assayed. “Our technique is much more accurate in determining both the size change and load of that mutation, whether it’s present in 50% or 5% or even fewer of the cells.”
Treatment typically suppresses cancer below detectable levels, but invariable it grows resistant to the therapy and “the earlier you can catch that relapse the better,” says Reed. The ability to detect small amounts of a mutant before it creates a medical problem is central to both early detection and ongoing cancer management.
Rearrangement mutations may involve several different classes of events—including deletions, duplications, inversions, and translocations—which fundamentally change the structure of DNA, says Reed. The new technique is specific to detecting those involving a change in size, a “common mechanism” in many cancers and neurological diseases caused by various biological processes, including DNA replication and DNA repair.
Using the novel approach, the cost of each digital PCR reaction costs less than a dollar to scan, Reed says. Most comparable assays run in the “tens to hundreds of dollars.” Hundreds, if not thousands, could be spent trying to detect length polymorphism via DNA sequencing since multiple rounds of testing and results analysis would be needed.
“The method we’re using … doesn’t really have any more complexity than a PCR assay itself,” he continues. “It can easily be done by most lab technicians.” The outlay for both materials and analysis is low, and the ordering oncologist or neurologist gets better information.
The next step for the VCU research team is to conduct a diagnostic trial that employs more patients and a broader scope of mutations, says Reed. Discussions are underway with several potential partners interested in the novel assay, which VCU could potentially out-licensed to a trial-sponsoring diagnostics company. Academic spinouts are also a possibility, although no commercialization efforts have yet to launch.