Efficient detection of the V600E mutation of the BRAF gene in papillary thyroid carcinoma using multiplex allele-specific polymerase chain reaction combined with denaturing high-performance liquid chromatography

Abstract

ABSTRACT. Mutations in the V-raf murine sarcoma viral oncogene homolog B1 gene (BRAF) play an important role in the pathogenesis of papillary thyroid cancer (PTC). In this study, a BRAF V600E mutation was detected in formalin-fixed and paraffin-embedded PTC samples using multiplex allele-specific polymerase chain reaction and denaturing high-performance liquid chromatography. The sensitivity of the assays was validated in two cell lines, K1 and RO82-W-1, which were respectively positive and negative for the mutation. The method enabled detection of very low concentrations Efficient detection of BRAF V600E mutation in thyroid cancer (~1%) of the mutation, with positive findings obtained in 119 of the 187 samples (63.6%). The mutation was not detected in 20 cases of benign thyroid diseases. The presence of the mutation was significantly associated with ages >45 years, the tumor-nodesmetastasis stage, region of metastasis, and clinical outcome (P < 0.05). Although no correlation was found between the mutation and lymph node metastasis, the proportions of patients carrying a mutation differed significantly between central and lateral cervical lymph node metastasis (P < 0.05). Multivariate logistic regression analysis also indicated the presence of mutation, tumor size, and cervical lymph node metastasis to be independent predictors for recurrence and distant metastasis. We conclude that a high proportion of PTC cases likely harbors the BRAF V600E mutation. This mutation can be used as an independent factor for predicting the recurrence and distal metastasis of PTC tumors.

Can detection of Braf p.V600E mutation be improved? Comparison of allele specific multiplex sequencing to present tests

“…[4,5] In attempting to meet these objectives, methods with significantly increased sensitivities and specificities have been developed, including allele-specific PCR. [6][7][8][9][10][11][12][13] Although allele-specific PCR and ASMS both use allelespecific primers, they are two quite different methods. In allele-specific PCR, allele-specific primers are used in the amplification step.…”