BACKGROUND: KRAS mutation testing is recommended for the discernment of metastatic colorectal cancer patients who are unlikely to benefit from anti-epidermal growth factor receptor antibodies. A recently developed amplification refractory mutation-Scorpion system is becoming a standard method for KRAS mutant detection. The feasibility and robustness of this system using DNA samples from clinically available formalin-fixed, paraffin-embedded specimens were evaluated. METHODS: Genomic DNA from macro-dissected 110 specimens was applied for the KRAS mutant detection using a commercial amplification refractory mutation-Scorpion system kit. Success rate and mutant detection rate of the test were evaluated. RESULTS: Small intra- and inter-lot deviations of the testing kit and a good concordance among different real-time polymerase chain reaction systems suggested the reliability of the amplification refractory mutation-Scorpion system. Though one-third of the 110 samples that were tested did not contain a sufficient amount of DNA to detect a 1% concentration of mutant alleles, the mutant detection rate was not impaired using tumor DNA concentrated by macro-dissection. Using a higher amount of template DNA, which supposedly contained abundant interfering substances, prevented the detection of the exogenous control amplicons, resulting in a reduced success rate. Adjusting the template amount according to the total DNA concentration might reduce the failure rate. CONCLUSION: The amplification refractory mutation-Scorpion system with formalin-fixed, paraffin-embedded specimen-derived DNA samples exhibited an acceptable feasibility and robustness suitable for routine clinical practice.
BACKGROUND:KRAS mutation testing is recommended for the discernment of metastatic colorectal cancerpatients who are unlikely to benefit from anti-epidermal growth factor receptor antibodies. A recently developed amplification refractory mutation-Scorpion system is becoming a standard method for KRAS mutant detection. The feasibility and robustness of this system using DNA samples from clinically available formalin-fixed, paraffin-embedded specimens were evaluated. METHODS: Genomic DNA from macro-dissected 110 specimens was applied for the KRAS mutant detection using a commercial amplification refractory mutation-Scorpion system kit. Success rate and mutant detection rate of the test were evaluated. RESULTS: Small intra- and inter-lot deviations of the testing kit and a good concordance among different real-time polymerase chain reaction systems suggested the reliability of the amplification refractory mutation-Scorpion system. Though one-third of the 110 samples that were tested did not contain a sufficient amount of DNA to detect a 1% concentration of mutant alleles, the mutant detection rate was not impaired using tumor DNA concentrated by macro-dissection. Using a higher amount of template DNA, which supposedly contained abundant interfering substances, prevented the detection of the exogenous control amplicons, resulting in a reduced success rate. Adjusting the template amount according to the total DNA concentration might reduce the failure rate. CONCLUSION: The amplification refractory mutation-Scorpion system with formalin-fixed, paraffin-embedded specimen-derived DNA samples exhibited an acceptable feasibility and robustness suitable for routine clinical practice.
Authors: H Bando; T Yoshino; K Tsuchihara; N Ogasawara; N Fuse; T Kojima; M Tahara; M Kojima; K Kaneko; T Doi; A Ochiai; H Esumi; A Ohtsu Journal: Br J Cancer Date: 2011-07-05 Impact factor: 7.640