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Biased discordance of KRAS mutation detection in archived colorectal cancer specimens between the ARMS-Scorpion
Hideaki Bando1, Katsuya Tsuchihara, Takayuki Yoshino
1Cancer Physiology Project, National Cancer Center Hospital East, Kashiwa, Chiba 277-8577, Japan.
Objective:
The concordance of KRAS mutation detection between the amplification refractory mutation system-Scorpion assay and direct sequencing was evaluated with clinically available formalin-fixed, paraffin-embedded specimens of metastatic colorectal cancers.
Methods:
Genomic DNA from 120 macrodissected specimens was examined by the amplification refractory mutation system-Scorpion assay and direct sequencing. DNA mixtures of wild-type and mutant KRAS genes were prepared from the peripheral blood and the SW620 human colon cancer cell line for the model experiments.
Results:
KRAS mutation was identified in 50 samples (41.7%) by the amplification refractory mutation system-Scorpion assay and 42 samples (35.0%) by direct sequencing. Discordance between the two methods was observed for samples with smaller amounts of amplifiable DNA. The sensitivity of direct sequencing was impaired by the decrease in template DNA and polymerase chain reaction cycles in the experimental models.
Conclusions:
Decreased sensitivity of direct sequencing caused by insufficient polymerase chain reaction amplification resulted in biased discordance between direct sequencing and amplification refractory mutation system-Scorpion. Polymerase chain reaction conditions satisfactory for amplifying tens of haploid copies of genomic DNA to the saturation level might be necessary to ensure the robustness of the direct sequencing-based method employed for formalin-fixed, paraffin-embedded specimen-derived DNA samples.
Insights
The amplification refractory mutation system-Scorpion assay showed higher concordance for KRAS mutation detection in metastatic colorectal cancer specimens compared to direct sequencing, especially with limited DNA. Optimized PCR is crucial for direct sequencing accuracy.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- KRAS mutations are key drivers in metastatic colorectal cancer (mCRC).
- Accurate detection of KRAS mutations is critical for guiding targeted therapy in mCRC patients.
- Formalin-fixed, paraffin-embedded (FFPE) specimens are commonly used but present DNA quality challenges.
Purpose of the Study:
- To evaluate the concordance of KRAS mutation detection between the amplification refractory mutation system-Scorpion (ARMS-Scorpion) assay and direct sequencing.
- To assess the performance of these methods using clinically available FFPE specimens of mCRC.
- To identify factors influencing discordance between the two molecular diagnostic techniques.
Main Methods:
- Genomic DNA was extracted from 120 macrodissected FFPE mCRC specimens.
- KRAS mutation status was determined using both the ARMS-Scorpion assay and Sanger direct sequencing.
- Experimental models with varying amounts of wild-type and mutant KRAS DNA were used to assess sensitivity.
Main Results:
- The ARMS-Scorpion assay detected KRAS mutations in 41.7% (50/120) of samples, while direct sequencing identified mutations in 35.0% (42/120).
- Discordance was more frequent in samples with lower amounts of amplifiable DNA.
- Direct sequencing sensitivity was compromised by reduced template DNA and fewer PCR cycles in experimental models.
Conclusions:
- Insufficient polymerase chain reaction (PCR) amplification in direct sequencing leads to decreased sensitivity and biased discordance with the ARMS-Scorpion assay.
- Robust direct sequencing of FFPE-derived DNA requires optimized PCR conditions to achieve sufficient amplification of genomic DNA.
- The ARMS-Scorpion assay demonstrates higher reliability for KRAS mutation detection in challenging FFPE samples.
