November 15, 2017 (Vol. 37, No. 20)

Handheld System Detects Single Molecules, Measures ctDNA in Blood and Urine

The cost of DNA sequencing has dropped to a point where researchers and clinicians are eyeing liquid biopsies that analyze circulating tumor DNA (ctDNA) as an attractive way to monitor cancer progression. Liquid biopsies could replace traditional solid biopsies, which are invasive, costly, painful, and sometimes dangerous.

The benefits of measuring ctDNA in patients’ blood or urine specimens are clear, but the uptake of ctDNA detection technology has been hampered by the technology’s high costs (~$2,500 per test) and long processing times (currently two to four weeks). These limitations affect the number of tests that can be completed and the amount of data that can be collected, prolonging and increasing the costs of clinical trials that must be completed before a given test can be cleared by the FDA. Indeed, only a few liquid biopsy tests have been approved to date.

Liquid biopsy systems have the potential to increase testing rates and reduce the costs of clinical trials—as well as the prices of individual tests. This potential will not be realized, however, unless liquid biopsy systems offer simplicity, economy, and point-of-use convenience in addition to the ability to detect clinically relevant levels of ctDNA from blood and urine samples.

Liquid biopsy systems possessing the requisite attributes will not only bring more and better tests to market faster and at lower costs, they will also allow testing to be conducted in a doctor’s office, or even in a patient’s home, expediting clinical decisions and courses of treatment.

For example, if a patient’s cancer were to return, necessitating a change in therapy, the recurrence could be detected within days or weeks, rather than months, potentially extending the patient’s lifespan and quality of life, while savings tens or hundreds of thousands of dollars in drug costs for a therapy that’s no longer effective. Further, the cost of liquid biopsy testing could drop to tens or hundreds of dollars, rather than thousands.

Handheld Liquid Biopsy Testing Kits

Two Pore Guys (2PG) has developed a novel point-of-use molecular diagnostics platform that has shown promise for liquid biopsy testing. Unlike biological nanopores used in some DNA sequencing technologies, 2PG’s system uses silicon nanopore chips to detect single molecules, one by one, like grains of sand passing through an hourglass.

When a negatively charged molecule, such as DNA, enters the pore along its transition, it impedes the current, which is continuously measured. The amount of current shift represents the level of impedance, which is proportional to the molecule’s bulk, or thickness, which could be accentuated by secondary structure. The time the molecule takes to pass through the pore is often governed by the length of the molecule, although other molecular features can cause drag (such as the degree to which the molecule interacts with the pore or binds with positively charged chemical species). As each molecule in the chamber passes through the pore and is measured, this “event” is plotted on a 2D graph (Figure 1).

The time and the impedance measurements are used to produce event signatures that reflect the nature of the molecule. More interestingly, one can alter the molecules to create desired signatures (Figure 2).

Figure 1. Nanopore sensors enable the analysis of single-molecule “events.”

The molecular structures depicted in Figure 2 carry single-nucleotide polymer (SNP)-specific probes. One probe binds to the wild-type KRAS gene, and another probe binds to the G12D point mutation. To create distinct populations on the current-impedance plot, one probe was tagged with a PEG linker, and the other probe was tagged with two PEG linkers. PEGs provide bulk, so when they pass through the pores, both the depth and duration of the current impedance are affected.

Because molecules reach equilibrium within a fluidic buffer, only a small fraction of the total samples need to be tested to achieve 99.9% confidence that a particular target is present in the entire matrix. If molecules are rare, the test can be run longer to achieve the desired confidence values. One can also add more nanopore chips to count more molecules per unit time.

Researchers and assay developers have used the 2PG platform to develop rapid tests for pathogens, field-based agricultural targets, proteins (such as antibodies and toxins), and even individual elements (such as lead, in water-monitoring applications).

Assay developers can adapt reagents from existing diagnostic tests for use on the platform, such as the primers that amplify ctDNA in a liquid biopsy test. The only proprietary modification of these reagents is the linking of PEGylated probes that hybridize directly with the specific DNA sequence of interest. (Locked nucleic acids, bridged nucleic acids, and many other probes may be used.)

Figure 2. Sequence-specific probe payloads enable multiplexed SNP detection.

Pilot Study at UCSF

Andrew Ko, M.D., professor of clinical medicine at the University of California, San Francisco (UCSF), conducted a pilot study to determine the sensitivity and accuracy of the platform for liquid biopsy. He presented his results at the Cambridge Healthtech Institute’s 2017 Next Generation Dx Summit. According to Dr. Ko, 2PG’s platform detected the KRAS G12D point mutation from ctDNA isolated in both blood and urine samples at clinically actionable levels.

For this study, samples were taken from nine patients who had colorectal or pancreatic cancer. The patient’s tumors were previously confirmed via DNA sequencing of biopsied tissue known to harbor the mutation. Blood samples were provided by all patients, and urine samples were also available from five of the patients.

The KRAS G12D mutation was detected in all nine blood samples, and more importantly, in all five urine samples, demonstrating the viability of a small, low-cost system that could eliminate the need for blood draws. Even more significantly, the process directly determined the mutant allele frequency, which may prove to be essential for clinical use in determining therapeutic treatment. Comparison studies to predicate platform quantitative polymerase chain reaction assays were performed, and similar results were obtained.

This represents the first time a handheld testing system was used in a clinical liquid biopsy test and demonstrates a key potential use of the platform: enabling low-cost, practical serial monitoring to measure how mutation status changes over time. Such a system could vastly improve the data collection necessary for FDA approval of new liquid biopsy tests. It could also promote the development of testing solutions that would be both economically viable and effective in settings beyond the laboratory, that is, in clinics around the globe and even in people’s homes.

Broad Applications for Handheld Molecular Detection

2PG provides a platform that is intended to help independent developers create assays that can detect virtually any kind of molecule. This is akin to the smartphone model, where a universal device supports third-party products and services for diverse industries. Early adopters of the platform include diagnostic, drug, and agricultural companies looking to expand their existing markets into the point-of-use market by leveraging their IP portfolio of reagents.

2PG’s devices may also contribute to the “Internet of things” by wirelessly transmitting anonymized data to authorized systems and cloud infrastructures. This kind of functionality would ameliorate the “last mile” problem particular to the accumulation and exchange of electronic health records. Essentially, the problem is that diagnostic test results are still mostly manually entered by lab operators into legacy systems.

Possible future applications for 2PG assays include telemedicine, home testing, clinical trials for drug development, and global tracking of pathogens and diseases. Non-human testing examples include the monitoring of food processing plants (to detect listeria and other pathogens), farms (to detect crop-destroying viruses), animals (to detect infections), and environmental reservoirs (to detect cholera in rivers or disease vectors in mosquito traps). Monsanto recently announced that it intends to evaluate the technology and develop assays for the detection of certain biomolecules in crops, pests, and pathogens.

2PG’s battery-operated device allows for nearly universal global accessibility and can be deployed in many clinical or applied testing situations, even where conditions are challenging, as they often are in remote regions or anywhere electrical grids are unreliable.

Dan Heller ([email protected]) is chief executive officer of Two Pore Guys.

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