Content » Vol 96, Issue 1

Short communication

Melanoma with BRAF Mutation in Circulating Cell-free DNA despite no Mutation in the Primary Lesion: A Case Report

Atsuko Ashida, Hisashi Uhara, Asuka Mikoshiba, Kaori Sakaizawa, Naomi Kumagai, Hiroshi Koga and Ryuhei Okuyama*

Department of Dermatology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan. *E-mail:

Accepted Jun 25, 2015; Epub ahead of print Jun 30, 2015

Substantial advances have been made recently in the treatment of metastatic melanoma, with the advent of therapies such as BRAF inhibitors (iBRAF), which have been shown to be effective in the treatment of BRAF mutation-positive melanoma (1). The frequency of BRAF mutations is 50–60% in Caucasians and 20–30% in Asian populations (1–3). Mutation is usually determined by Sanger sequencing and high-sensitivity PCR-based methods, such as the Cobas 4800 BRAF Mutation Test (Roche Molecular Diagnostics, Basel, Switzerland), using primary or metastatic lesions. However, the accuracy of these tests is grossly influenced by the quality and quantity of the DNA extracted from the tissues. Thus, BRAF mutation can be missed in cases of contamination, widespread necrosis, or when only small quantities of DNA are available. We describe here a case of advanced-stage melanoma with a BRAFV600E mutation. Although the BRAFV600E mutation was not detected in the DNA of the primary lesion, it was detected in circulating cell-free DNA (cfDNA) in peripheral blood. cfDNA includes DNA from apoptotic and necrotic cancer cells, it harbours the genetic alterations present in the tumour (4, 5), and is useful for evaluating tumour features in patients with advanced cancer.


A 47-year-old Japanese woman was diagnosed with stage IV melanoma. She had a history of limb pain for 3 months, and computed tomography (CT) scans showed multiple bone, lung and lymph nodes metastases. A pigmented macule, measuring 39×14 mm, with extensive regression, was situated on the right abdomen. Histopathologically, she was diagnosed with melanoma. Although tumour cells were positive for S-100, MART-1 and HMB45, the lesion contained < 10% tumour cells with numerous pigment-laden macrophages. After obtaining written informed consent, we analysed the tumour for BRAF mutation using the Sanger method (3), but did not detect BRAF mutation in the primary lesion (Fig. 1a). We also performed Competitive Allele-Specific TaqMan PCR (castPCR: Life Technologies, Carlsbad, CA, USA) for BRAFV600E, which has higher specificity than Sanger sequencing. Assay sensitivity was established by the manufacturer at 0.1%. However, castPCR amplified neither BRAFV600E nor BRAFwild from DNA of the primary lesion, suggesting that the abundant melanin in the tumour cells, which is known to inhibit PCR, interfered with the reaction (Fig. 1b). Since it was impractical to take biopsies of the metastatic lesions, we analysed cfDNA for the BRAF mutation. DNA was extracted from plasma and serum using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Venlo, The Netherlands), and castPCR was performed using DNA from the plasma and serum. The results showed that BRAFV600E comprised 63.2% and 49.8% of the total BRAF isolated from plasma and serum, respectively (Fig. 1c, d). In addition, no BRAF mutation was detected in samples from 5 healthy volunteers. Unfortunately, the general condition of the patient declined rapidly, and she died before iBRAF could be administered.


Fig. 1. (a) BRAF mutation analysis of the primary lesion using Sanger sequencing. The arrow indicates the wild-type profile. Three bases, GTG, encode the amino acid V at codon 600. (b) BRAF mutation analysis of the primary lesion using castPCR. Representative amplification of DNA from the primary lesion using BRAFwild-type and BRAFV600E probes. Analysis was performed in triplicate, and samples with a quantification cycle > 37 were considered non-informative. Neither V600E-mutated nor wild-type curves crossed the threshold level (black line). BRAF mutation analysis of cfDNA derived from (c) plasma and (d) serum using Competitive Allele-Specific TaqMan PCR (castPCR: Life Technologies, Carlsbad, CA, USA). Patient samples were measured in triplicate. V600E mutated curves and wild-type curves crossed the threshold level (arrows). Mutation analysis was repeated 3 times, independently. MT: mutated-type; WT: wild-type.


Apoptotic and necrotic cancer cells are a key source of cfDNA, harbouring the same genetic alterations present in the primary tumour (4). Detection of BRAF mutations in blood could represent a useful biomarker for non-invasive diagnosis. However, a crucial issue is that blood usually contains only a small amount of tumour-derived DNA. Most PCR methods developed to detect BRAFV600E are limited by the presence of a high proportion of wild-type BRAF that interferes with cfDNA mutation assays. Several methods, including BEAMing (Beads, Emulsions, Amplification, and Magnetics) (6), droplet digital PCR (7), and next-generation sequencing (8), have been developed to facilitate mutation analysis from cfDNA in a highly specific and sensitive manner. However, these methods require specialized equipment. castPCR requires no specific instruments except a real-time PCR system, and is able to detect and quantitate rare mutations in samples containing large amounts of wild-type DNA. The high sensitivity of this method suggests that analysis of cfDNA isolated from blood for the BRAFV600E mutation could help to identify those patients with melanoma who would benefit from iBRAF therapy.

The case described here shows that cfDNA is useful for identifying mutations in melanoma with regression, in which melanoma cells have disappeared or become reduced in number and have been replaced by pigment-laden macrophages with inflammatory infiltration. Regression in primary melanoma occurs in 10–35% of cases (9). Sanger analysis requires a tumour cell content of at least 40–50% to produce accurate results (10); analysis using samples with lower numbers of tumour cells can result in a failure to detect BRAF mutation and lead to inadequate treatment. In summary, in cases of melanoma with extensive regression, it may be beneficial to assess BRAF mutations in the blood as well as in the primary lesion.


This study was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese government (AA).

The authors declare no conflicts of interest.


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