Published in:
01-10-2016 | Original Article
Quantitative analysis of the BRAF V600E mutation in circulating tumor-derived DNA in melanoma patients using competitive allele-specific TaqMan PCR
Published in: International Journal of Clinical Oncology | Issue 5/2016
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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.