Abstract. There is currently no standard method for the detection of Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation status in colorectal tumors. In the present study, we compared the KRAS mutation detection ability of four methods: direct sequencing, Scorpion-ARMS assaying, pyrosequencing and multi-analyte profiling (Luminex xMAP). We evaluated 73 cases of metastatic colorectal cancer (mCRC) resistant to irinotecan, oxaliplatin and fluoropyrimidine that were enrolled in an all-case study of cetuximab. The KRAS mutation detection capacity of the four analytical methods was compared using DNA samples extracted from tumor tissue, and the detection success rate and concordance of the detection results were evaluated. KRAS mutations were detected by direct sequencing, Scorpion-ARMS assays, pyrosequencing and Luminex xMAP at success rates of 93.2%, 97.3%, 95.9% and 94.5%, respectively. The concordance rates of the detection results by Scorpion-ARMS, pyrosequencing and Luminex xMAP with those of direct sequencing were 0.897, 0.923 and 0.900 (κ statistics), respectively. The direct sequencing method could not determine KRAS mutation status in five DNA samples. Of these, Scorpion-ARMS, pyrosequencing and Luminex xMAP successfully detected three, two and one KRAS mutation statuses, respectively. Three cases demonstrated inconsistent results, whereby Luminex xMAP detected mutated KRAS in two samples while wild-type KRAS was detected by the other methods. In the remaining case, direct sequencing detected wild-type KRAS, which was identified as mutated KRAS by the other methods. In conclusion, we confirmed that Scorpion-ARMS, pyrosequencing and Luminex xMAP were equally reliable in detecting KRAS mutation status in mCRC. However, in rare cases, the KRAS status was differentially diagnosed using these methods.
IntroductionCetuximab is a monoclonal antibody that targets the extracellular domain of the epidermal growth factor receptor (EGFR), and is an essential treatment option in patients with metastatic colorectal cancer (mCRC). Numerous researchers have reported that anti-EGFR agents have extremely poor antitumor effects in chemotherapy for mCRC with mutated Kirsten rat sarcoma viral oncogene homolog (KRAS) (1-5), providing clear evidence that administration of anti-EGFR agents is recommended only for mCRC with wild-type KRAS. However, although a number of methods may be used for KRAS mutation testing with varying sensitivity and specificity levels, no standard method has yet been recommended for clinical practice. Therefore, the use of these detection assays is somewhat erratic worldwide.In Japan, cetuximab was administered for ~18 months following its launch in September 2009 without determination of KRAS mutation status, since the above-mentioned analytical methods were not covered by health insurance. The direct sequencing method (6) was covered in April 2010, followed by multi-analyte profiling (Luminex xMAP) technology (7) in March 2011 and Scorpion-ARMS assays (8)