Abstract
Microarray technology fails in detecting point mutations present in a small fraction of cells from heterogeneous tissue samples or in plasma in a background of wild-type cell-free circulating tumor DNA (ctDNA). The aim of this study is to overcome the lack of sensitivity and specificity of current microarray approaches introducing a rapid and sensitive microarray-based assay for the multiplex detection of minority mutations of oncogenes (KRAS, NRAS and BRAF) with relevant diagnostics implications in tissue biopsies and plasma samples in metastatic colorectal cancer patients. In our approach, either wild-type or mutated PCR fragments are hybridized in solution, in a temperature gradient, with a set of reporters with a 5' domain, complementary to the target sequences and a 3' domain complementary to a surface immobilized probe. Upon specific hybridization in solution, which occurs specifically thanks to the temperature gradients, wild-type and mutated samples are captured at specific location on the surface by hybridization of the 3’ reporter domain with its complementary immobilized probe sequence. The most common mutations in KRAS, NRAS and BRAF genes were detected in less than 90 minutes in tissue biopsies and plasma samples of metastatic colorectal cancer patients. Moreover, the method was able to reveal mutant alleles representing less than 0,3% of total DNA. We demonstrated detection limits superior to those provided by many current technologies in the detection of RAS and BRAF gene superfamily mutations, a level of sensitivity compatible with the analysis of cell free circulating tumor DNA in liquid biopsy.
Highlights
The identification of DNA variants that can cause diseases is a central aim in human genetics
The discovery that tumor cells release DNA fragments in blood, urine or other body fluid samples, paves the way to a paradigm shift in cancer diagnostics introducing the concept of liquid biopsy: a term that refers to a novel, non-invasive technique used for detecting cancer biomarkers [3,4]. circulating tumor DNA (ctDNA) belongs to the pool of the total circulating cell free-DNA in blood
In an effort to overcome the limitations of both existing approaches, we introduce an innovative assay for the simultaneous detection of single mutations in different oncogenes, based on microarray technology combined with solution hybridization
Summary
The identification of DNA variants that can cause diseases is a central aim in human genetics. CtDNA provides real-time molecular information allowing monitoring treatment response and relapsing as it contains genetic alteration of both primary and metastatic lesions, such as point mutations, copy number variations and insertions/deletions [5,6]. Detecting mutation in ctDNA is challenging since the lower number of mutant copies of cancer origin are masked by the large amount of wild-type DNA mostly from contaminant leukocytes [7]. With ddPCR only the genes that are the most susceptible to mutations are analyzed, at first, giving the patient the choice of whether to pursue additional tests based on the results. The downside of this approach to testing is it is time and cost consuming
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