Breaking the Sensitivity Barrier, Digital PCR Technology Offers Absolute Quantification
Pierre Laurent-Puig and collaborators use a new dPCR platform to track circulating tumor DNA, finding rare mutations that were previously undetectable.
When Pierre Laurent-Puig evaluates a new technology platform, he doesn’t just think about how it could work in a research lab. A trained gastroenterologist who focuses on genetic alterations in cancer, Laurent-Puig’s focus is all about improving his ability to help patients.
So when Laurent-Puig and his collaborators had a chance to perform studies on the droplet-based digital PCR technology underlying RainDance’s new RainDrop instrument, he targeted the area that had the most potential for making a difference in the lives of cancer patients: improving detection for cell-free circulating DNA in blood, stool, and other samples.
This free circulating tumor DNA is especially important to Laurent-Puig, who wants to use it to track critical biomarkers that could be used for diagnostic or prognostic purposes — and could offer a much better, noninvasive alternative to current techniques for identifying patients whose colon cancer has metastasized.
But with standard approaches, such as using PCR assays with TaqMan probes, Laurent-Puig’s ability to detect tumoral DNA was limited to cells present in at least 0.1 percent to 1 percent of the sample. Cells that were more rare than that could slip by unnoticed — a critical failing for a diagnostic approach to patient care. “I would like to have an absolute quantification of the mutant in plasma or blood samples,” Laurent-Puig says. When it comes to detecting these mutations, “it’s not important to say ‘yes’ or ‘no’ — what is important is to say the quantification, and then to develop a threshold that has relevance for patients with cancer.”
The microdroplet-based digital PCR platform under development at RainDance changed that dramatically. In a recent Lab on a Chip paper from Laurent-Puig and his collaborators, the team used the digital PCR technology and demonstrated the ability to detect a single mutated copy of KRAS in a background of 200,000 wild-type KRAS copies. By processing reactions in millions of picoliter droplets, the technology improves sensitivity by two orders of magnitude, Laurent-Puig says. “You have absolute quantification,” he adds. “You can evaluate the proportion of circulating tumor DNA as compared to non-tumor DNA.”
That means the old sensitivity limit of 1 percent or so no longer stands in the way of Laurent-Puig’s finding the tumoral DNA he’s after. “With this method, the limitation is no longer a technical limitation. It’s now a limitation of what amount of DNA you can test,” he says.
In the same Lab on a Chip paper, Laurent-Puig and his teammates were able to screen for seven different KRAS mutations in parallel. As the sensitivity limit shifts from a technical one to one based on DNA volume, that parallel screening will be essential for finding as much information as possible from as little sample as possible.
Going forward, Laurent-Puig aims to use the RainDance digital PCR technology on samples from a cohort of 200 colon cancer patients, followed by DNA sequencing of the tumors that are detected. “We will try to determine the prognostic impact of the presence of tumor circulating DNA in patients with colon cancer stage 2 and 3,” he says. “We will determine the mutation in the tumors — KRAS, BRAF, P53, and so on — and use that to build a personalized follow-up based on the genetic characterization of the tumor.”
— Meredith Salisbury