DNA, Hold the Cells
Sometimes information from a malignancy is necessary for diagnosis and treatment, but for a host of reasons it’s not convenient or even possible to biopsy the tumor. In such cases researchers can try to identify and assay circulating tumor cells, or perhaps look for biomarkers found in circulating cell-free DNA (CF-DNA).
Pamela Pinzani, Ph.D., and her colleagues at the University of Florence knew that the BRAFV600E mutation was an early event in most melanomas, and they wanted to find a way to exploit that to create an assay for diagnosis and prediction of response to therapy.
To find the mutation, they needed to see a difference from wild type of a single base in a minute amount of DNA. Tumor DNA (i.e., BRAF-mutated DNA) typically accounts for around 1–10% total CF-DNA, which, in turn, is typically found in the order of nanograms per mL of plasma.
A standard real-time PCR assay—the “technique of choice”—turned out not to be specific enough. So they tried to increase the specificity by incorporating locked nucleic acid bases into the mutation-specific probe in place of some normal DNA bases, conferring a higher avidity.
Yet this, too, was “not sufficient by itself to increase specificity to the level we needed,” Dr. Pinzani recalled. They next turned to a primer that was specific for the mutation, increasing the avidity and efficiency of the amplification reaction of the mutated allele and simultaneously decreasing them for the wild-type sequence. The team was thus able to obtain a standard curve and use this to measure BRAFV600E in plasma samples.
Most melanoma patients were found to have high levels of BRAFV600E mutation, with a statistical difference between the healthy individuals and melanoma patients. They are now trying to compare it with some other circulating biomarkers.
“I suppose that more than one circulating biomarker is necessary to have 100% specificity in these patients,” she said. “Because there are some melanomas that do not show this variant, we’ll have to use a multiparameter approach.” Fortunately, the experience with BRAFV600E should provide an excellent place to begin looking for other melanoma-specific markers.