Quantitative analysis of the BRAF V600E mutation in circulating tumor-derived DNA in melanoma patients using competitive allele-specific TaqMan PCR

Abstract

Background

BRAF ^V600E is a common mutation in melanoma, and BRAF inhibitors are effective in treating of BRAF mutation-positive melanoma. DNA carrying this mutation is released from melanoma cells into the circulation. As such, circulating tumor-derived DNA (ctDNA) in peripheral blood represents a novel biomarker for evaluating tumor features in cancer patients. However, ctDNA is present in the peripheral blood at very low levels, which makes the detection of specific mutations in this DNA a challenge. Competitive allele-specific TaqMan PCR (castPCR), a straightforward commercially available assay, is a sensitive technique for quantitating a small amount of DNA.

Methods

The level of BRAF ^V600E ctDNA was quantified by castPCR in 26 consecutive plasma samples from six melanoma patients.

Results

The castPCR assay was performed using a mixture of BRAF ^V600E DNA and BRAF ^wild DNA and found to be able to detect BRAF ^V600E at a fractional abundance of ≥0.5 % in 2- to 10-ng samples of genomic DNA. Cell-free DNA was then extracted from peripheral blood samples collected from six patients with melanoma harboring the BRAF ^V600E mutation. BRAF ^V600E ctDNA was detected in three patients, at a fractional abundance of between 1.28 and 58.0 % of total BRAF cell-free DNA. The abundance of BRAF ^V600E ctDNA correlated with tumor burden, as determined by computed tomography imaging. In two cases, an increase in the level of BRAF ^V600E ctDNA preceded exacerbation of clinical symptoms.

Conclusion

The castPCR assay can detect and quantitate small amounts of BRAF ^V600E ctDNA in samples containing large amounts of BRAF ^wild cell-free DNA. Thus, we suggest that the castPCR assay is suitable for monitoring ctDNA in the plasma of melanoma patients.

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