Sensitive detection of BRAF V600E mutation by Amplification Refractory Mutation System (ARMS)-PCR

Abstract

BackgroundBRAF mutations occur in approximately 8% of all human cancers and approach 50% in melanoma and papillary carcinoma of thyroid. These mutations provide potentially valuable diagnostic, prognostic and treatment response prediction markers. A sensitive, specific, low-cost assay to detect these mutations is needed.ResultsTo detect BRAF V600E mutation in formalin-fixed, paraffin-embedded (FFPE) tissue, we developed a method using Amplification Refractory Mutation System (ARMS)-PCR. This method was designed to amplify three products in a single reaction tube: a 200 bp common product serving as an amplification control, a 144 bp BRAF V600E specific product, and a 97 bp wild-type (wt) specific product. The sensitivity of this method was determined to be as low as 0.5% for the BRAF V600E allele in a wild-type background. This method was successfully validated in 72 thyroid tumors. It detected V600E mutation in 22 out of 33 (67%) of the conventional papillary thyroid carcinoma (PTC), 8 out of 12 (75%) of the tall-cell variant of PTC, whereas none of the 10 follicular variant of PTC showed BRAF V600E mutation. In addition, none of the 14 follicular adenomas and 3 follicular carcinomas had BRAF V600E mutation. As a comparison method, direct dideoxy sequencing found only 27 out of 30 (90%) mutations detected by ARMS-PCR method, suggesting that this ARMS-PCR method has higher sensitivity.ConclusionsOur ARMS-PCR method provides a new tool for rapid detection of BRAF V600E mutation. Our results indicate that ARMS-PCR is more sensitive than automated dideoxy sequencing in detecting low BRAF V600E allele burdens in FFPE tumor specimen. The strategy of this ARMS-PCR design may be adapted for early detection of point mutations of a variety of biomarker genes.

Usefulness of immunohistochemistry for the detection of the BRAF V600E mutation in ovarian serous borderline tumors

“…Notably, intra-and inter-tumor heterogeneity of BRAF V600E mutations in melanomas has also been reported by the group (21). Amplification refractory mutation system (ARMS)-PCR detected BRAF V600E in only 55% (30/55) cases of papillary thyroid carcinoma, whereas Sas detected the mutation in 27 of 30 cases (22). we selected CAST-PCR, a technology based on small-fragment amplification and qPCR suitable for low quantities of DNA templates (16).…”

FCGR3A158V/F Polymorphism and Response to Frontline R-CHOP Therapy in Diffuse Large B-Cell Lymphoma

“…The FCGR3A gene polymorphism was detected by polymerase chain reaction (PCR)-sequencing assay as previously described (Huang et al, 2013). The first PCR primers for the FCGR3A gene SNP at locus 158 were 5-ATA TTT ACA GAA TGG CAC AGG-3 and 5-GAC TTG GTA CCC AGG TTG , while the second PCR primers for the FCGR3A gene at locus 158 were 5-ATA TTT ACA GAA TGG CAC AGG-3 and 5-ATG CTG CAG AGT GAA TGA CAC-3, generating a 394-bp fragment.…”

Tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) rapidly identified a critical missense mutation (P236T) of bovine ACADVL gene affecting growth traits

“…Studies have shown that T-ARMS-PCR assay is more sensitive than the automated dideoxy method at low allele frequencies (Huang et al, 2013;Medrano and de Oliveira, 2014;Singh et al, 2014). The main principle of the T-ARMS-PCR method is that Taq DNA polymerase lacks the 3′ → 5′exonuclease activity, and if the primer's 3′ end is mismatched hence, its amplification rate is slower or it fails.…”