Evaluation of the Anyplex BRAF V600E real-time detection assay using dual-priming oligonucleotide technology in fine-needle aspirates of thyroid nodules.
BACKGROUND
Several molecular assays have been developed to detect the BRAF V600E mutation in fine needle aspirates (FNAs) for the diagnosis of papillary thyroid cancer. Using a multiplex PCR technique, we evaluated the Anyplex BRAF V600E Real-time Detection (Anyplex) assay and compared its efficacy with that of the Seeplex BRAF V600E ACE Detection (Seeplex) method.


METHODS
We tested 258 consecutive FNA specimens using the Seeplex and Anyplex assays. Any conflicting results between the two assays were confirmed by using mutant enrichment with 3'-modified oligonucleotide (MEMO) sequencing. The limits of detection (LODs) and reproducibility for each assay were evaluated with serially diluted DNA from a BRAF V600E-positive cell line.


RESULTS
The BRAF V600E mutation was detected in 36.4% (94/258) FNA specimens by either the Seeplex or Anyplex assay. Results for the two assays showed 93.4% (241/258) agreement, with a kappa value of 0.861 (95% confidence interval, 0.798-0.923). Of the eight specimens that were BRAF V600E-positive by the Anyplex assay but not by the Seeplex assay, five were found to be BRAF V600E-positive by MEMO sequencing. The mutation detection rate of the Seeplex and Anyplex assays was 79.0% and 84.0%, respectively, in the FNA specimens diagnosed as malignant (n=81). The LOD as determined by probit analysis was 0.046% (95% confidence interval, 0.019-0.532%).


CONCLUSIONS
The Anyplex assay performed better than the Seeplex assay with respect to the detection of the BRAF V600E mutation.
Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Rihwa
R
Choi
Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Kyung Sun
KS
Park
Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Jong Won
JW
Kim
Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea. changski@skku.edu.
Chang Seok
CS
Ki
D000328:Adult; D000368:Aged; D044466:Asian People / Q000235:genetics; D044963:Biopsy, Fine-Needle; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction; D009841:Oligonucleotides / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D056910:Republic of Korea; D016606:Thyroid Nodule / Q000378:metabolism* / Q000473:pathology
D017931:DNA Primers; D009841:Oligonucleotides; D004247:DNA; D048493:Proto-Oncogene Proteins B-raf
Anyplex; BRAF V600E; Evaluation; Fine-needle aspiration; Real-time PCR; Seeplex