Abstract
The high degree of intra-tumor heterogeneity has meant that it is important to develop sensitive and selective assays to detect low-abundance KRAS mutations in metastatic colorectal carcinoma (mCRC) patients. As a major potential source of tumor DNA in the aforementioned genotyping assays, it was necessary to conduct an analysis on both the quality and quantity of DNA extracted from formalin-fixed paraffin-embedded (FFPE). Therefore, four commercial FFPE DNA extraction kits were initially compared with respect to their ability to facilitate extraction of amplifiable DNA. The results showed that TrimGen kits showed the greatest performance in relation to the quality and quantity of extracted FFPE DNA solutions. Using DNA extracted by TrimGen kits as a template for tumor genotyping, a real-time wild-type blocking PCR (WTB-PCR) assay was subsequently developed to detect the aforementioned KRAS mutations in mCRC patients. The results showed that WTB-PCR facilitated the detection of mutated alleles at a ratio of 1:10,000 (i.e. 0.01%) wild-type alleles. When the assay was subsequently used to test 49 mCRC patients, the results showed that the mutation detection levels of the WTB-PCR assay (61.8%; 30/49) were significantly higher than that of traditional PCR (38.8%; 19/49). Following the use of the real-time WTB-PCR assay, the ΔC q method was used to quantitatively analyze the mutation levels associated with KRAS in each FFPE sample. The results showed that the mutant levels ranged from 53.74 to 0.12% in the patients analyzed. In conclusion, the current real-time WTB-PCR is a rapid, simple, and low-cost method that permits the detection of trace amounts of the mutated KRAS gene.
Highlights
Human colorectal carcinoma (CRC) is one of the most common malignancies in worldwide countries including China
Comparative analysis of four commercially available formalin-fixed paraffin-embedded (FFPE) DNA extraction kits The characteristics of DNA extracted from FFPE sections can affect the accuracy associated with results generated from genotyping assays
For the 20 samples scraped from the glass slide, the average amount of extracted DNA was 2670 ng, 1910 ng, 660 ng, and 365 ng for TrimGen, Epicentre, Gene Tech, and Qiagen kits, respectively (Fig 2a and 2b)
Summary
Human colorectal carcinoma (CRC) is one of the most common malignancies in worldwide countries including China. Cetuximab and panitumumab are two approved monoclonal antibody-based therapeutic medicines that target the epidermal growth factor receptor (EGFR). These therapeutic agents have been used for the palliative treatment of human metastatic CRC (mCRC) since 2004 and 2007, respectively. A recent study indicated that a higher-sensitivity KRAS mutation analysis method could help to identify patients who had poor responses to anti-EGFR antibody therapy in mCRC [9,10,11,12]. The development of reliable and sensitive methods to detect low-abundance mutations associated with KRAS would be extremely useful determinants prior to the clinical application of anti-EGFR antibody therapies in mCRC
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