PCR-Based Liquid Biopsy Is a Simple and Sensitive Cancer Diagnostic

This figure illustrates how the assay can be used clinically. In this patient, the data enabled the investigators to count the number of DNA molecules containing the KRAS G12V mutation and, therefore, count the number of molecules that came from the patient’s tumor. The green dots represent molecules found to contain the mutation sequence, and the blue dots represent wild-type normal DNA molecules. The information was derived from less than a nanogram of cell-free DNA. [Hanlee P. Ji/Stanford Genome Technology Center]

Noninvasive cancer diagnostics are rapidly becoming an essential part of precision medicine therapies. These cutting-edge assays are not only useful in the preliminary diagnosis of various cancerous conditions but also help physicians track the progression of cancers during and after treatment. Typically, these tests have employed the use of next-generation sequencing (NGS) techniques; while they are sensitive, they are also complicated and costly. Now, a team of investigators from Stanford University has just released findings from their work on a new type of test that can detect genetic mutations in minute amounts of DNA released from cancer cells into the blood using a relatively simple PCR based assay.  

The results from this study—published today in The Journal of Molecular Diagnostics in an article entitled “Single-Color Digital PCR Provides High-Performance Detection of Cancer Mutations from Circulating DNA”—show that this highly sensitive test has the potential to be personalized to recognize mutations unique to any individual cancer. The test, which the researchers dubbed single-color digital PCR, requires only a fraction of a tube of blood and can detect as few as three mutation-bearing molecules in a single reaction.

“For monitoring patient tumors, only a handful of blood tests are available that are limited to only several types of cancers,” explained senior study investigator Hanlee Ji, M.D., associate professor in the department of medicine at Stanford University and senior associate director of the Stanford Genome Technology Center. “Nearly all cancer patients require monitoring by whole-body imaging, which can be costly, complex, and time-consuming.”

In the new study, the researchers described the use of the test to analyze samples from six patients. Of these patients, five were previously diagnosed with colorectal cancer and one with cholangiocarcinoma. The research team was able to identify tumor-derived circulating DNA from three out of six patients. In one patient, the assay showed the presence of three different mutations. The three patients, whose samples did not show elevated cancer DNA, were undergoing active treatment at the time of collection.

“Molecular tests like the one we have developed will enable patients to be monitored at every visit, and thus have the potential for quickly tracking cancer growth and spread,” Dr. Li noted. “Moreover, the test's rapid turnaround and relatively low cost, especially compared to next-generation DNA sequencing, provide a potential opportunity for universal monitoring of more patients than is currently done.”

The single-color digital PCR test offers several advantages over other methods of circulating tumor DNA analysis, compared to next-generation targeted sequencing and fluorescent probe-based digital PCR assays. The main advantage is that the new technique does not rely on preamplification, which can introduce errors and biases.

“This approach relied on a double-stranded DNA intercalator dye and paired allele-specific DNA primer sets to determine an absolute count of both the mutation and wild-type–bearing DNA molecules present in the sample,” the authors wrote. “The cell-free DNA assay uses an input of 1 ng of nonamplified DNA, approximately 300 genome equivalents, and has a molecular limit of detection of three mutation DNA genome-equivalent molecules per assay reaction. When using more genome equivalents as input, we demonstrated a sensitivity of 0.10% for detecting the BRAF V600E and KRAS G12D mutations. We developed several mutation assays specific to the cancer driver mutations of patients' tumors and detected these same mutations directly from the nonamplified, circulating cell-free DNA.”

The investigators are optimistic about their finding and look forward to testing their assay on larger sample populations of cancer patients. Due to the speed of the assay and its reduced costs, the research team is hopeful that their test could be employed in an array of physician’s offices and clinics for routine cancer monitoring.  

“This test is simple enough to set up and analyze without extensive training, and, therefore, it can be implemented by anyone, making it highly accessible to any laboratory,” concluded lead study author Christina Wood-Bouwens, a research professional at Stanford University School of Medicine. “It has been truly motivating to work with a technology that will help transform the way that we monitor and treat individuals with cancer. I am excited to share our findings with the cancer research community.”