Biased discordance of KRAS mutation detection in archived colorectal cancer specimens between the ARMS-Scorpion method and direct sequencing.
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.
Cancer Physiology Project, National Cancer Center Hospital East, Kashiwa, Chiba 277-8577, Japan.
Hideaki
H
Bando
Katsuya
K
Tsuchihara
Takayuki
T
Yoshino
Motohiro
M
Kojima
Naomi
N
Ogasawara
Hiraku
H
Fukushima
Atsushi
A
Ochiai
Atsushi
A
Ohtsu
Hiroyasu
H
Esumi
D015982:Bias; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D004252:DNA Mutational Analysis / Q000379:methods*; D005557:Formaldehyde; D006801:Humans; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D018631:ras Proteins / Q000235:genetics*
C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D005557:Formaldehyde; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins