An experimental test successfully detected cancerous mutations in tumor cells by analyzing their DNA that is free-floating in blood, employing a strip test that displays results within 10 minutes.
In a paper published April 12 in Analytica Chimica Acta, the researchers said their liquid biopsy test is less invasive than standard tissue sampling and could be expanded to detect other cancerous genes.
Like many healthy cells, cells from cancerous tumors can release fragments of their DNA into the bloodstream when they die. The genetic code of these cells contains mutations that promote cancerous growth, and the blood-borne DNA fragments — called circulating tumor DNA — have become a popular research subject for new cancer diagnosis, treatment and prognosis.
Tissue biopsy, the removal and testing of a tissue sample for the presence of tumors or other diseases, is the "gold standard" for cancer diagnosis, according to a 2016 review paper in Oncotarget, although it has some limitations that include "unsatisfactory" detection of early-stage tumors or residual lesions.
The review said that drawing and analyzing blood, the most common form of liquid biopsy, stand to be a promising alternative because of the highly sensitive and specific results of screening for circulating tumor DNA, although the technique still needs further testing for regular clinical use.
Circulating tumor DNA, or ctDNA, is "a very promising noninvasive biomarker allowing early cancer detection and monitoring of cancer-specific aberrations at all times during the course of the disease, bypassing the limitations of tumor biopsies," said Despina Kalogianni, an assistant professor of chemistry at the University of Patras and the new study's senior author.
The test was demonstrated to detect different variants of the KRAS gene, which can be associated with colon cancer, depending on its mutations. Kalogianni and her co-authors looked for the standard variant and three point mutations in circulating tumor DNA in blood samples of three colon-cancer patients and three healthy donors.
The researchers treated test strips with a compound based on gold nanoparticles, which creates a red spot on the strips if a given gene variant is present. Microscopic bits of gold were connected to the protein streptavidin, which binds very strongly to biotin, or vitamin B₇.
Meanwhile, the KRAS genes were treated with specially designed primer molecules in four primer extension reactions, binding an identifying "flag" to each of the DNA fragments. The primer molecules were then bound to biotin, which the gold-nanoparticle compounds latch onto.
Four strip tests were used for each sample, one for each KRAS variant. In principle, the gold-nanoparticle compounds would create a red spot only if the variant being tested were present.
The test successfully detected each of the KRAS variants, and the results were verified by analyzing tissue samples from the patients. The red spots appeared within 10 minutes of applying the samples to the strip tests, and the entire process took between 3 and 3 1/2 hours, the researchers reported.
"The developed lateral flow assay offers a rapid diagnostic strip test for visual genotyping of ctDNA as an alternative approach for liquid biopsy applications," Kalogianni said.
Unlike many other existing techniques, according to the chemist, the new test is simple and quick and does not require specialized technology or personnel to read the results — the red spots on the strips, in this case.
"It is also cost-effective, with high detectability, specificity and reproducibility and is a portable and universal device that can be applied for the genotyping of any ctDNA or gene of interest," Kalogianni said.
She and her lab are planning to develop a rapid strip test that detects multiple mutations in a single strip. They also want to target more mutations and cancer-related genes, she said.
The study, "Liquid biopsy genotyping by a simple lateral flow strip assay with visual detection," published April 12 in Analytica Chimica Acta, was authored by Panagiota Kalligosfyri, Sofia Nikou, Vasiliki Bravou and Despina Kalogianni, University of Patras.