In this study,
BRAF V600mut ctDNA was detected at baseline in 75 % of stage IV melanoma patients with a known
BRAF V600 mutation. This is in line with two other studies using PCR based methods, where
BRAF V600mut ctDNA was detected in plasma of 73–84 % of
BRAF V600 mutant melanoma patients [
6,
25]. These results are also in line with an overall detection rate of 77 % in 4 clinical studies with 746 stage IV
BRAF V600 mutant melanoma patients using BEAMing (beads, emulsions, amplification, and magnetics analysis) after cfDNA extraction from plasma [
26‐
30]. Moreover, one study showed a 100 % agreement between digital PCR, qPCR and BEAMing, suggesting that the suboptimal detection rate of
BRAF V600 mutant DNA in plasma in advanced melanoma is the result of a variable amount of
BRAF V600mut ctDNA in advanced melanoma patients, rather than an variability in the sensitivity of different test-platforms [
25]. We observed that after initiating BRAF/MEK inhibitor treatment,
BRAF V600mut ctDNA can drop below the detection limit, notwithstanding the absence of radiological complete remission. This phenomenon of rapid decrease in the concentration of
BRAF V600mut ctDNA has been reported before and has been attributed to the destruction of tumor cells and a subsequent rapid clearance of ctDNA [
6,
31‐
33]. Our combined observations of (1) decrease of the
BRAF V600mut ctDNA concentration within days after treatment initiation, (2) of early increase during disease progression and (3) of early increase after discontinuation of targeted therapy, are suggestive of a correlation between ctDNA levels and the proliferation of melanoma cells. Therefore
BRAF V600mut ctDNA seems to reflect the
BRAF V600mut-dependent
proliferative tumor burden, and not tumor mass as evidenced by CT-imaging. From this perspective, ctDNA could be attributed to increased cell death during melanoma cell proliferation or might be actively secreted or passively released by living cells, rather than by apoptotic or necrotic cells [
34]. This latter hypothesis is supported by several preclinical arguments. Spontaneous active DNA release or apoptosis have been put forward as the main source of ctDNA, because plasma DNA often presents a ladder pattern, typical for active cleaving, when subjected to electrophoresis [
32,
35]. Several arguments support the hypothesis that DNA is primarily released by living cancer cells rather than by apoptotic cancer cells: (1) DNA concentration increases in normal lymphocyte cultures following stimulation with phytohemagglutinin, lipopolysaccharide or antigen; (2) in a leukemic cell-line malignant cells released newly synthesized DNA; (3) cancer cell DNA concentration in cell culture supernatant increases with cell proliferation when few apoptotic or necrotic cells are present; and (4) circulating exosomal
BRAF V600E mutant DNA was isolated from SK-MEL-28 melanoma-bearing mice [
32,
33,
36,
37].
In our study population, PFS was significantly better for the patients in whom
BRAF V600mut ctDNA became undetectable after the initiation of targeted therapy. When the
BRAF V600mut ctDNA fraction increased during treatment, clinical PD always followed within 2 months. Moreover, in 44 % of cases an increase in the mutant fraction preceded PD. We recently reported that reappearance of
BRAF V600mut ctDNA in plasma cfDNA can precede changes on PET–CT in patients under BRAF/MEK inhibitor treatment [
34]. In this study an increase in the
BRAF V600mut ctDNA fraction preceded PD with a median interval of 25 days. However, plasma samples were collected on a monthly basis, whereas imaging was performed every 2 months. In a study on ctDNA monitoring in metastatic breast cancer, increasing levels of ctDNA appeared on average 5 months before the establishment of PD by means of imaging [
3].
An increase in the
BRAF V600mut ctDNA fraction during BRAF/MEK targeted therapy could potentially serve as a trigger for early evaluation with imaging techniques, and allow for a timely switch to immunotherapy or other appropriate therapeutic interventions (e.g. stereotactic radiotherapy to brain metastases). Early detection of progression is important in metastatic melanoma, because of the aggressive course of the disease as exemplified by the observation that the central nervous system is a frequent site (30 %) of first progression under BRAF inhibitor therapy [
17]. Moreover, potential second line therapeutic options such as ipilimumab and, to a lesser extent anti-PD-1 therapies, are associated with a latency of their anti-tumor effect. Therefore, the possibility of offering immunotherapy may be compromised if PD is only detected on imaging or when clinical symptoms are present [
19,
20].