Rapid detection of low-abundance K-ras mutation in stools of colorectal cancer patients using chip-based temperature gradient capillary electrophoresis

Abstract

Mutant K-ras provides an independent negative predictive marker for epidermal growth factor receptor (EGFR)-targeted therapy in colorectal cancers (CRCs). Rapid, sensitive, and cost-effective screening for K-ras status will overarch rational personalized medicine. Stool-based DNA testing offers unique advantages for CRC screening such as noninvasiveness, high specificity, and patient compliance, whereas complicated procedures and the low sensitivity of the present approaches have hampered its application on a wide scale. In this study, a chip-based temperature gradient capillary electrophoresis (TGCE) technique was applied to detect low-abundance K-ras mutations under a pooled experiment and analyze K-ras mutations in 30 paired stool samples and cancer tissues of CRC patients and 15 stool samples of healthy volunteers. The chip-based TGCE results showed that the successful analysis of K-ras status could be achieved within 6 min with an extremely low sample consumption of 14 nl. Detection is sensitive enough to reliably report 0.2% mutant CRC cells in a wild-type background, and 0.5 ng of template DNA was sufficient for chip-based TGCE. Of the 30 stool samples of CRC patients analyzed, 17 (57%) harbored K-ras mutations, and the lowest percentage of the detectable mutant K-ras in stool samples was 2%. The coincidence rate for K-ras mutations between stools and tissues obtained by the chip-based method reached 97% (29/30). One of the 15 stool samples of normal controls carried K-ras mutations, producing a specificity of 93%. Clone sequencing data entirely confirmed the results obtained by chip-based TGCE. The study demonstrates that chip-based TGCE is capable of rapidly screening low-abundance K-ras mutations with high sensitivity, reproducibility, simplicity, and significant savings of time and sample. Application of this method to genotype the K-ras gene in stools would provide a potential means for predicting the effectiveness of EGFR-targeted therapy in CRC patients using noninvasive approaches.

Detection of Low-Abundance KRAS Mutations in Colorectal Cancer Using Microfluidic Capillary Electrophoresis-Based Restriction Fragment Length Polymorphism Method with Optimized Assay Conditions

“…Moreover, considering cancer-derived samples often consist of highly heterogeneous mixtures of stromal cells and cancer cells, lower sensitivity of sequencing analysis (20%) tends to cause false negative results and misconduct the clinical therapy [12]. Consequently, a variety of more sensitive PCR-based screening techniques have been developed, and indicated that methods with high sensitivities could achieve higher mutation detection rate, which will in turn improve the CRC treatment [13]–[15]. Because only limited hotspot mutations in KRAS codons 12 and 13 are proved to be related to the clinical medicine [16], the simple and cost-effective PCR/restriction fragment length polymorphism (RFLP) analysis has been extensively used to detect KRAS mutations [17]–[20], which allows the reliable discrimination between wild-type sequences and homozygous or heterozygous point mutations by generating or destructing restriction sites through PCR and subsequent electrophoresis [21].…”

Cell-free nucleic acids as noninvasive biomarkers for colorectal cancer detection

“…In several research studies using small cohorts (<100), it was an overall concordance between tissue and stool for Kras genotype and it was possible to detect mutation even in 1000-fold excess of wild-type Kras (Mixich et al, 2007). The overall results showed that Kras mutations has 34–87.5% of sensitivity for detecting CRC patients and the specificity was very high, reaching in some studies 100% (Dong et al, 2001; Doolittle et al, 2001; Rengucci et al, 2001; Calistri et al, 2003; Chien et al, 2007; Mixich et al, 2007; Zhang et al, 2011a). …”

Microfluidics technology: future prospects for molecular diagnostics

“…Zhang et al 53 developed a microchip-based temperature gradient CE system for the detection of low-abundance K-ras mutation, a biomarker for the epidermal growth factor receptor-targeted therapy in CRCs. The microfluidic chip was fabricated with glass and had a cross-channel design with sample loading and separation channels of 1.0 and 4.5 cm, respectively.…”