When it comes to liquid biopsy, not all tests are equal. In fact, many suffer from challenges, such as low sensitivity, expensive equipment requirements, or the need for error-prone enhancement steps like pre-amplification. Can these obstacles be overcome? Certainly – and a new polymerase chain reaction technique known as single-color digital PCR can help. The assay requires very little blood, carries a price tag much lower than sophisticated sequencing methods, and bypasses the pre-amplification step and its concomitant risk of error. It’s possible that this technique, and others like it, may offer a new avenue for lower-cost, higher-accuracy liquid biopsy.
Why develop a new type of liquid biopsy?
Current methods of liquid biopsy, such as next generation sequencing and probe-based digital PCR, can be time-consuming and costly to optimize and run. Single-color digital PCR, which is essentially a standard PCR reaction, is cheap, simple to optimize and implement, and can be completed in a matter of hours after sample collection.
Tracking a patient’s tumor burden is a critically important part of their medical management. Given the high cost and complexity of imaging studies, these scans are performed at intervals of months at a time – or even longer. The clear need for a faster, more frequent means of monitoring inspired us to use digital PCR to create a simple, low-cost method of monitoring cancer growth and spread so that it can be implemented at every visit.
How does single-color digital PCR work?
Digital PCR involves partitioning one or several DNA molecules into individual micro-volume droplet reactions that each generate a specific PCR amplicon product. Single-color digital PCR uses double-strand DNA binding dye, which incorporates into amplicon products and produces a fluorescent signal. We use this signal to distinguish between droplets that contain the amplicon of interest and those that are empty. By counting positive droplets, we can easily count the number of molecules that contain the target DNA, even if those molecules are incredibly rare within the sample. This approach enables users to design standard PCR primers for any target of interest to obtain absolute quantification in a matter of hours.
Because this blood-based test is exquisitely sensitive, capable of detecting tiny amounts of cancer DNA, it is possible to apply the technology to a wide range of patients. We require only a fraction of a tube of blood to check for tumor DNA, and the test can be completed in a matter of hours. Most importantly, the test can be “personalized” to detect mutations unique to any individual cancer. As a result, this molecular test can be universally applied to monitor cancer in any patient, regardless of the type of tumor.
Any surprises along the way?
One of the most surprising results we have encountered is the variability in concentration of cell-free DNA across samples. It can often be a two- to 100-fold difference! We find dramatic differences in cell-free DNA between different patients, as well as between individual patient time points. This speaks to the importance of developing highly sensitive tests capable of handling low-input amounts while still providing informative results.
How will this change routine laboratory testing?
The ideal clinical test would be low-cost, high-performance, delivered within 12 hours, and require only limited amounts of blood. Single-color digital PCR addresses many of these points.
There are only a handful of blood tests for monitoring patients’ tumors at the moment, and those are limited to only a few types of cancers. Because there are so few liquid biopsy options, nearly all cancer patients require whole-body imaging, such as CT scans, to ascertain the presence and growth of their disease. Our new liquid biopsy method will enable laboratory medical professionals to monitor disease status without the need for complex, invasive, or time-consuming tests. Instead, a single tube of blood – or less – is sufficient to check for tumor DNA molecules. And it’s so simple that any laboratory professional can prepare and perform the analysis without extensive training.
What are your top tips for liquid biopsy?
As the liquid biopsy research space expands, it is important to continue developing tools and technologies that are customizable, sensitive, and low-cost. These features are critical in generating real-time information that can affect patient care and medical treatment.
As a practicing medical oncologist, I see many patient scenarios where liquid biopsy would be informative for the patient. Such rapid, robust tests could become commonplace in clinical practice in a matter of years – and will definitely be the go-to for cancer testing in the future.
Hanlee Ji is Associate Professor of Medicine (Oncology) at Stanford University, Stanford, USA.
Hanlee Ji is Associate Professor of Medicine (Oncology) at Stanford University, Stanford, USA.
