Researchers have developed a sensitive method to directly detect mutations in the BRAF gene without amplifying a DNA or RNA sample. Professor Christoph Gerber of the Swiss Nanoscience Institute at the University of Basel, Switzerland; Donata Rimoldi of the Ludwig Institute for Cancer Research in Lausanne, Switzerland; and their respective colleagues developed a nanotechnology approach to detect the mutation in RNA from melanoma cells with the help of a microscopic cantilever.
“The method is based on specific binding of molecules to the top surface of a microscopic cantilever,” says Francois Huber of the Swiss Nanoscience Institute, who is first author of the Nature Nanotechnology report. “For specific detection, the cantilevers are first coated with a layer of DNA molecules, which can bind mutated RNA from cells.” RNA extracted from cancer tissue samples or tissue cultures can be used directly. Binding of the RNA to the anchored DNA deflects the cantilever and the degree of bending is measured with a laser beam. The researchers demonstrated that their new tool can distinguish cells carrying a BRAF mutation from those with a wild-type BRAF gene.
The major advantage of the technique, according to Huber, is that it does not depend on labeling of DNA or RNA molecules or on amplification of the molecules, which can introduce artifacts and distort results. “One potential disadvantage is that the molecular interactions need to take place very close to the surface of the cantilever to generate a substantial surface stress,” Huber says.
Approximately 50% of metastatic melanoma tumors harbor a mutation in amino acid 600 of the BRAF gene. Over 90% of these BRAF mutations are V600E, a single base pair change from a thymine to an adenine.
Currently, these BRAF mutations are detected using a polymerase chain reaction (PCR) assay. Researchers extract DNA from a tissue sample and use PCR to amplify the mutated BRAF gene. In the U.S., the FDA-approved cobas® 4800 BRAF V600 Mutation test is widely used to analyze the BRAF mutation status of melanoma patients and guide treatment decisions. This PCR test is performed on formalin-fixed, paraffin-embedded tumor tissue samples.
But the microcantilever approach could be an easier and faster way to detect mutations from clinical tumor samples, according to the researchers. It requires only a small amount of RNA, and without the need for amplification, the method could shorten the time interval between patient sample processing and treatment.
While a previous proof-of-principle study showed that direct RNA and DNA detection was possible with the microcantilever, this was the first demonstration of the ability of the device to detect a single point mutation in a background of unrelated sequences of total cellular RNA.
The new method can even be used to analyze several mutations at once using an array of cantilevers. In theory, it is also possible that novel mutations could be discovered with the microcantilever method, Huber says, but the location of the target within a transcribed RNA must be known.
Huber and colleagues are now validating the technology using patient samples. “Extended studies are necessary for commercialization and clinical applications,” says Huber, who is working to improve detection statistics for the device. “Ultimately we would like to see the application of nanotechnology and particularly our method in the clinic.”
The team is also working on technology that can capture circulating tumor cells (CTCs) from a blood sample (seeCancer Commons blog post: Circulating Tumor Cells May Help Determine Melanoma Prognosis for further information on CTC assays). This technique is not invasive and would circumvent the need for a tumor biopsy, allowing easier testing for patients with inaccessible tumors such as those found in the pancreas, liver, and lungs.