Allele specific Taqman-based real-time PCR assay to quantify circulating BRAFV600E mutated DNA in plasma of melanoma patients

Abstract

BRAF is the most frequently mutated oncogene in melanoma with BRAF(V600E) mutation accounting for 92% of all BRAF variants. As this event occurs early in melanoma progression, the quantification of BRAF-mutated alleles in plasma may represent a useful biomarker for noninvasive diagnosis and prediction of response to therapy. We propose an assay based on the use of a locked nucleic acid probe and an allele specific primer to measure plasma-circulating BRAF(V600E) concentration in patients affected by cutaneous melanoma (n=55) and non-melanoma skin cancers (n=13) as well as 18 healthy subjects. The assay is highly sensitive and accurate in detecting down to 0.3% of mutated allele in plasma. A significant difference between the control group and invasive melanomas (p<0.01) was evidenced in BRAF(V600E) concentration, either as relative percentage or absolute values. ROC curve indicated that BRAF(V600E) absolute concentration has the maximal diagnostic relevance with 97% sensitivity and 83% specificity. Comparison of the results obtained in plasma with those found in the corresponding tissues indicated an 80% concordance. The allele specific Taqman-based real-time PCR assay allows the sensitive, accurate and reliable measurement of BRAF(V600E) mutated DNA in plasma.