Evaluation of the Anyplex BRAF V600E Real-Time Detection Assay Using Dual-Priming Oligonucleotide Technology in Fine-Needle Aspirates of Thyroid Nodules

Abstract

BackgroundSeveral 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.MethodsWe 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.ResultsThe 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%).ConclusionsThe Anyplex assay performed better than the Seeplex assay with respect to the detection of the BRAF V600E mutation.

Evaluation of Droplet Digital PCR for the Detection of BRAF V600E in Fine-Needle Aspiration Specimens of Thyroid Nodules

“…In contrast, the risk of malignancy of indeterminate thyroid nodules, such as atypia of undetermined significance/follicular lesion of undermined significance (AUS/FLUS) and follicular neoplasm/suspicious for follicular neoplasm (FN/SFN), is 11%-23% and 20%-29%, respectively [11]. Therefore, additional molecular assays are used to investigate these indeterminate nodules [10,[12][13][14].…”

Reply:

“…Several molecular assays have been developed to detect the B-Raf proto-oncogene (BRAF) V600E mutation in fine-needle aspirates for the diagnosis of papillary thyroid cancer (PTC). 13 Musholt et al 14 considered that mutations of RET/PTC, RAS, and PAX8/peroxisome proliferator-activated receptor ␥ (PPAR␥) were predominantly associated with thyroid malignancy with varying frequency and had less impact on the clinical management. However, in the study of Song et al, 15 BRAF mutations were the most common ones observed in PTCs, followed by RET/PTC rearrangements and RAS mutations, while follicular thyroid cancers were more likely to have RAS mutations or PAX8/PPAR␥ rearrangements.…”