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Feasibility and robustness of amplification refractory mutation system (ARMS)-based KRAS testing using clinically
Naomi Ogasawara1, Hideaki Bando, Yasuyuki Kawamoto
1Cancer Physiology Project, Research Center for Innovative Oncology, National Cancer Center Hospital East, Kashiwa, Japan.
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.
Insights
The amplification refractory mutation-Scorpion system reliably detects KRAS mutations in colorectal cancer patients using formalin-fixed, paraffin-embedded specimens. This method is feasible and robust for routine clinical practice.
Area of Science:
- Molecular diagnostics
- Oncology
- Genetics
Background:
- KRAS mutation testing is crucial for identifying metastatic colorectal cancer patients unlikely to respond to anti-EGFR therapies.
- The amplification refractory mutation-Scorpion system offers a new method for KRAS mutant detection.
- Evaluating the system's performance with formalin-fixed, paraffin-embedded (FFPE) DNA is essential for clinical application.
Purpose of the Study:
- To assess the feasibility and robustness of the amplification refractory mutation-Scorpion system.
- To determine the system's effectiveness using DNA extracted from FFPE specimens.
Main Methods:
- Genomic DNA was extracted from 110 macro-dissected FFPE specimens.
- KRAS mutant detection was performed using a commercial amplification refractory mutation-Scorpion system kit.
- Success and mutant detection rates were evaluated.
Main Results:
- The amplification refractory mutation-Scorpion system demonstrated small lot-to-lot variations and good concordance across real-time PCR platforms, indicating reliability.
- Despite some samples lacking sufficient DNA for 1% mutant allele detection, macro-dissection preserved mutant detection rates.
- Optimizing template DNA concentration is key to improving success rates by avoiding interference from abundant substances.
Conclusions:
- The amplification refractory mutation-Scorpion system is feasible and robust for routine clinical use.
- FFPE-derived DNA is suitable for KRAS mutation testing with this system.
- The method supports personalized treatment decisions for colorectal cancer patients.
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