Small Samples; Big Promises

It’s hard to look at a laboratory medicine journal without seeing the words “liquid biopsy” these days. Small wonder the technique is such a hit – it’s simple, noninvasive, and makes use of emerging molecular techniques to tell us more than ever about the diseases our patients face. But with all of these advantages, liquid biopsy does face one challenge – sensitivity.

“The main issue with analyzing circulating cell-free DNA is that its concentration is low, and DNA of tumor origin is present at very low frequencies – sometimes only individual molecules,” says Anders Ståhlberg, docent in molecular medicine at the University of Gothenburg’s Sahlgrenska Cancer Center. “Standard techniques are not sensitive enough to find these rare molecules,” he continues, “but with new approaches such as our SiMSen-Seq technique, this is now possible.”

SiMSen-Seq allows the detection of circulating tumor DNA (ctDNA) in the blood with up to 1,000-fold more sensitivity than the methods currently in use(1)(2). Ståhlberg and his colleagues accomplish this feat by adding a molecular barcoding step. “In molecular DNA barcoding, a unique sequence is added to each individual DNA molecule that enables us to track all sequencing reads back to the original DNA molecule. By aligning reads with the same barcode, it is then possible to differentiate between true mutations and those resulting from polymerase errors.” SiMSEn-Seq is not the only liquid biopsy method to use barcoding, but Ståhlberg says that each method carries its own limitations. “Our contribution is that we managed to develop a cost-effective method that is simple to use, flexible to adjust, and can be used with minimal DNA input.”

What are the researchers doing with the technique now? Ståhlberg outlines a number of clinical investigations applying ultrasensitive mutation detection to liquid biopsy, including patients with childhood sarcomas, melanomas and breast cancers. He and his team are also applying their approach to areas beyond cancer, including chronic obstructive pulmonary disease and immunological responses. Nonetheless, he warns against jumping into liquid biopsy too fast. “The potential of circulating cell-free DNA is very high, but validation studies are important to prove its clinical value. You may find mutations without a disease – so we need to learn how and when to perform this type of analysis.”

Ståhlberg next plans to learn exactly which liquid biopsy applications gain the greatest clinical value from ultrasensitive mutation analysis. He and his colleagues have recently received funding from several collaborating organizations to start a translational genomics platform (3) working with liquid biopsies and ultrasensitive mutation analysis. And he’s optimistic about the future of liquid biopsy: “By analyzing patient-specific mutations in blood plasma, we anticipate improvements in diagnosis, treatment selection, prognosis, treatment monitoring and relapse detection.”