Abstract
In the era of personalized medicine, molecular profiling of patient tumors has become the standard practice, especially for patients with advanced disease. Activating point mutations of the KRAS proto-oncogene are clinically relevant for many types of cancer, including colorectal cancer (CRC). While several approaches have been developed for tumor genotyping, liquid biopsy has been gaining much attention in the clinical setting. Analysis of circulating tumor DNA for genetic alterations has been challenging, and many methodologies with both advantages and disadvantages have been developed. We here developed a gold nanoparticle-based rapid strip test that has been applied for the first time for the multiplex detection of KRAS mutations in circulating tumor DNA (ctDNA) of CRC patients. The method involved ctDNA isolation, PCR-amplification of the KRAS gene, multiplex primer extension (PEXT) reaction, and detection with a multiplex strip test. We have optimized the efficiency and specificity of the multiplex strip test in synthetic DNA targets, in colorectal cancer cell lines, in tissue samples, and in blood-derived ctDNA from patients with advanced colorectal cancer. The proposed strip test achieved rapid and easy multiplex detection (normal allele and three major single-point mutations) of the clinically relevant KRAS mutations in ctDNA in blood samples of CRC patients with high specificity and repeatability. This multiplex strip test represents a minimally invasive, rapid, low-cost, and promising diagnostic tool for the detection of clinically relevant mutations in cancer patients.
Highlights
Personalized medicine is based on recommendations according to genomic “drivers”of tumorigenesis [1]
We have developed a rapid multiplex strip test based on gold nanoparticles for the detection of gene mutations in circulating tumor DNA for non-invasive liquid biopsy applications
Optimization parameters involved samples that consisted of (i) four single-stranded synthetic DNA targets corresponded to the four KRAS alleles (Table 1) and DNA isolated from (ii) cell lines expressing the wild-type and the mutated (G12D) KRAS gene and (iii) formalin-fixed paraffin-embedded (FFPE) tissue samples from colorectal cancer (CRC)
Summary
Personalized medicine is based on recommendations according to genomic “drivers”of tumorigenesis [1]. Cancer develops through a multistage process, with the accumulation of somatic mutations and genetic alterations leading to increased cell proliferation and tumorigenesis [2]. Such mutations are known as “drivers” and are crucial for monitoring disease progression and resistance to targeted therapeutic agents [3,4]. Amongst known driver mutations, activating mutations in the KRAS or KRAS2 (Kirsten rat sarcoma virus 2 homolog) oncogene are found in several malignancies including colorectal cancer (CRC) [5,6,7,8]. KRAS mutations occur early in the development of colorectal cancer (CRC) and are strongly associated with resistance to therapies [10,11]. About 40% of CRC cases are KRAS-mutant-related, meaning that they cannot benefit from EGFR-targeted therapies [13]
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