The form of melanoma will be determined by a cantilever
Atomic force microscopy adapted for cancer diagnosis
Oleg Lischuk, N+1
Swiss scientists have developed a nanomechanical sensor for quick and easy diagnosis of the form of melanoma. The principle of its operation resembles an atomic force microscope. The report on the work is published in the journal Nano Letters (Huber et al., Fast Diagnostics of BRAF Mutations in Biopsies from Malignant Melanoma).
Melanoma is a malignant neoplasm of pigment cells (melanocytes). It is the most dangerous form of skin cancer, annually affecting more than 200 thousand and taking the lives of more than 50 thousand inhabitants of the Earth. In about half of the cases, the tumor cells carry a mutation of the BRAF gene, designated BRAFV600E. In recent years, biologics (such as vemurafenib) have been developed that selectively inhibit the mutant form of the protein. They prolong the life of patients more effectively and with fewer side effects than conventional chemotherapy, but their appointment requires an accurate diagnosis of the form of melanoma. Currently, BRAFV600E is detected by real-time biopsy PCR followed by DNA sequencing. Such diagnostics takes a lot of time, is expensive and often leads to false positive results.
To simplify the diagnosis of mutation, employees of the Universities of Basel and Lausanne have developed a sensor with a set of titanium-gold cantilevers (microelectromechanical probes), which are similar to those used in atomic force microscopy. On the surface of these probes, self-assembling layers of small single-stranded DNA of 18 nucleotides are covalently connected, which are complementary to the tumor RNA sites containing the desired mutation (RNA was chosen for diagnosis because there is more of it in the cell, and the RNA/DNA heterodimer exerts more pressure on the probe than the DNA/DNA homodimer). The control probes contain layers of oligonucleotides complementary to the usual form of BRAF, as well as non-specific sequences of polyadenosylcytosine.
When specially prepared RNA from the patient's biopsies is applied to the sensor, its mutated fragments are hybridized with the corresponding oligonucleotides. The emerging steric and ionic forces reject the cantilevers, and by the degree of this deviation, one can judge the presence of a mutant gene, its usual variant, or both of them (the same tumor may contain cells with different types of BRAF).
Experiments with cell lines have shown that the technique is able to detect BRAFV600E if its share is at least five percent of all copies of the gene in the sample. In a pilot clinical trial involving nine patients, the diagnostic system was able to identify mutant tumor samples with an accuracy not inferior to the traditional method. At the same time, PCR amplification or the use of labeled molecules are not required for the analysis, and both fresh and formalin- or paraffin-fixed tissue samples are suitable as a material.
The head of the work was Christoph Gerber, one of the developers of atomic force microscopy (for this he and his co–authors were awarded the Kavli Prize in Nanoscience in 2016). "Thirty years ago, we could not have foreseen that our technology would one day come to the clinic for the needs of personalized medicine," he said in a press release from Nanotechnology Supports Treatment of Malignant Melanoma.
The researchers note that the developed technique can serve to diagnose other tumors and mutations. To date, Gerber's laboratory has been able to use it to analyze the interaction of antigens with antibodies, DNA with transcription factors and antibiotics with bacteria. In addition, it was adapted to study the transcriptional activity of genes and the functions of transmembrane proteins.
Atomic force microscopy (a modification of scanning tunneling microscopy, created in 1982) allows using a cantilever to analyze the relief of surfaces with a resolution from nanometers to single atoms. This research method is widely used in physics, chemistry, biology and related disciplines.
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13.09.2016