CASE REPORT

Detection and Monitoring of the BRAF Mutation in

Circulating Tumor Cells and Circulating Tumor DNA in
BRAF-Mutated Lung Adenocarcinoma
Nicolas Guibert, MD,a,b Anne Pradines, PhD,b,c Anne Casanova, MS,b,c
Magali Farella, MS,b,c Laura Keller, PhD,b,c Jean-Charles Soria, MD, PhD,d
Gilles Favre, PhD,b,c Julien Mazires, MD, PhDa,b,*
a
Thoracic Oncology Department, Larrey Hospital, University Hospital of Toulouse, University of Toulouse III
(Paul Sabatier), Toulouse, France
b
Inserm, Centre de Recherche en Cancrologie de Toulouse, CRCT UMR-1037, Toulouse, France
c
Laboratoire de Biologie Mdicale Oncologique, Institut Universitaire du Cancer de Toulouse, France
d
Drug Development Department, Gustave Roussy Cancer Campus, Paris-Sud University, Villejuif, France

Received 2 May 2016; accepted 2 May 2016

Available online - 7 May 2016

Introduction mutation, a p.Arg132Cys-IDH1 mutation. For patients 2

The B-Raf proto-oncogene, serine/threonine kinase
gene (BRAF) V600E mutation occurs in less than 2% of
cases of nonsmall cell lung carcinoma (NSCLC); how-
ever, it has been associated with interesting response
rates to B-Raf proto-oncogene, serine/threonine kinase
(BRAF) (Minhibitors either alone or associated with
mitogen-activated protein kinase kinase (MEK) in-
hibitors.1 Cell-free circulating tumor DNA (cfDNA) and
circulating tumor cells (CTCs) have been described as
noninvasive tools to detect and monitor epidermal
growth factor receptor gene (EGFR) mutations in
NSCLC2,3 during cancer treatments but never for a BRAF
mutation. Moreover, no study has yet compared CTCs
and cfDNA for this purpose.
Case Reports
cfDNA and DNA extracted from CTCs obtained by
isolation according to size of epithelial tumor cells from
six patients treated for metastatic BRAF V600E NSCLC
were tested for the BRAF V600E mutation using digital
droplet polymerase chain reaction (PCR). This mutation
was detected in the cfDNA of all six patients but in the
CTCs of only one patient (Table 1).
In the rst of the six cases, the initial sample was
obtained at the time of resistance to the BRAF inhibitor.
Despite the addition of a MEK inhibitor, the patient suf-
fered disease progression. A dissociated plasma response
was then observed, with a decrease in the BRAF mutant
and an increase in BRAF wild type (WT) in cfDNA (Table 1
and Fig. 1). Targeted next-generation sequencing of the
biopsy specimen identied, besides the known BRAF
through 4 (Fig. 2), for whom rst blood samples were
obtained after failed chemotherapy and before initiation
of the BRAF inhibitor, we observed a good correlation
between variations in plasma BRAF mutants in cfDNA and
a response to BRAF inhibitors (Response Evaluation
Criteria in Solid Tumors 1.1 criteria).
Discussion
Rapid, noninvasive, and repeatable access to the
molecular prole of NSCLC is challenging. We herein
demonstrated the feasibility of detecting and monitoring
BRAF mutations in blood samples using digital droplet
PCR on a small number of patients. Of particular interest,
apart from in the intriguing rst case, the kinetics of
mutant cfDNA correlated well with changes in tumor
burden. These results are in agreement with another
report on BRAF-mutated melanoma.4 BRAF mutants in
cfDNA were often detected in small amounts, but no
positive droplets were detected in the WT samples,
indicating good specicity (see Table 1).
*Corresponding author.
Disclosure: The authors declare no conict of interest.
Address for correspondence: Julien Mazires, MD, PhD, Thoracic
Oncology Unit, Respiratory Disease Department, Hpital Larrey, CHU
Toulouse, Chemin de Pouvourville, 31059 Toulouse Cedex, France.
E-mail: mazieres.j@chu-toulouse.fr
2016 International Association for the Study of Lung Cancer.
Published by Elsevier Inc. All rights reserved.
ISSN: 1556-0864
http://dx.doi.org/10.1016/j.jtho.2016.05.001

Journal of Thoracic Oncology Vol. 11 No. 9: e109-e112

 e110 Guibert et al
Table 1. BRAF-Mutated and Wild-Type DNA in Plasma and CTCs during Treatment with BRAF Inhibitors
Positive
Last Treatment Mutant Negative Control Mutant Mutant/
Received (at Time CTCs/2 Copies/mL Control (10 ng DNA Copies/mL WT Copies/mL Total RECIST
Patient of Blood Collection) Spots in CTCs (WT Patients) A375) in ctDNA in ctDNA cfDNA Evaluation
1 Dabrafenib 1 0 0 145.9 600 590 50.4% DP
Dabrafenib trametinib 5 0 0 161.7 290 1250 18.8% DP
Dabrafenib trametinib 3 0 0 154.6 100 970 9.3%
2 Bevacizumab 3 0 0 149.6 27 1167 2.3% DP
Vemurafenib 3 0 0 146.3 274 27120 1%
3 Pemetrexed 0 1.6 0 152.8 1.6 350 0.45% PR
Vemurafenib 0 0 0 152.1 0 900 0% DP
Vemurafenib 0 0 0 146 5.8 400 1.4%
4 Pemetrexed 0 0 0 133.2 0.2 446 0.04% DP
Vemurafenib 0 0 0 149.8 1.4 1170 0.11%
5 Cisplatin pemetrexed 1 0.04 0 141.1 240 1460 14.1% DP
Vemurafenib NA NA NA NA NA NA NA

Journal of Thoracic Oncology

6 Pemetrexed 0 0.12 0 144.2 272 2815 8.8% NA
Vemurafenib NA NA NA NA NA NA NA
Note: Positive control: 10 ng DNA extracted from A375 cell line (BRAF V600Emutant melanoma cell line); Negative control: cfDNA extracted from plasma of patients with Kirsten rat viral sarcoma oncogene homolog
gene (KRAS)-mutated and BRAF WT lung adenocarcinomas.
BRAF, B-Raf proto-oncogene, serine/threonine kinase gene; BRAF, B-Raf proto-oncogene, serine/threonine kinase; CTCs, circulating tumor cells; WT, wild-type; ctDNA, circulating tumor DNA; cfDNA, circulating free
DNA; RECIST, Response Evaluation Criteria in Solid Tumors; DP, disease progression; PR, partial response; NA, not applicable.

Vol. 11 No. 9
September 2016 Monitoring Circulating BRAF in Lung Cancer e111

60 In patient 1, the decreased BRAF-mutated DNA indi-

cated that the BRAF inhibitor was still active on the BRAF
50
rao mutant copies/ WT clone. However, the concomitant increase in BRAF WT in
copies (%)
40 cfDNA and tumor progression suggests that this clone
was no longer predominant. The isocitrate dehydroge-
30 nase (NADP()), 1 systolic gene (IDH1) mutation confers
in vivo growth of the BRAF-mutated melanoma cell line5
20
and was probably the mechanism of resistance in this
10 case. No archival tissue was available to conrm that this
alteration was not initially present.
0
BRAFi + MEKi BRAFi + MEKi BRAFi + MEKi
Our observations suggest that plasma has better
sensitivity compared with CTCs. However, CTCs have
Figure 1. Variations in the ratio of B-Raf proto-oncogene, several advantages (prognostic value, uorescence in
serine/threonine kinase gene (BRAF)-mutated over wild- situ hybridization, immunocytochemistry) but are
type (WT) circulating free DNA during treatment with dab-
rafenib (a B-Raf proto-oncogene, serine/threonine kinase probably not as effective at detecting and monitoring
inhibitor [BRAFi]) and trametinib (a mitogen-activated pro- mutations.
tein kinase inhibitor [MEKi]) in patient 1 and the correlations In conclusion, analyses of BRAF mutants using digital
with a computed tomography scan. droplet PCR on cfDNA is feasible and appears to be more

Figure 2. The increase in B-Raf proto-oncogene, serine/threonine kinase gene (BRAF) V600Emutated circulating tumor-
specic DNA in patient 2 during treatment with vemurafenib concomitant with dramatic disease progression, as seen on a
computed tomography scan. The BRAF V600E probe plot is shown. The pink line is the threshold for positive versus negative
droplets.
e112 Guibert et al
sensitive than analyzing CTCs. This test could be useful
when following up BRAF-mutated lung adenocarcinoma.
References
1. Planchard D, Kim TM, Mazieres J, et al. Dabrafenib in
patients with BRAFV600E-positive advanced non-small-
cell lung cancer: a single-arm, multicentre, open-label,
phase 2 trial [e-pub ahead of print]. Lancet Oncol. 2016;
pii: S1470-2045(16)00077-2, accessed April 11, 2016.
2. Oxnard GR, Paweletz CP, Kuang Y, et al. Noninvasive
detection of response and resistance in EGFR-mutant lung
cancer using quantitative next-generation genotyping
Journal of Thoracic Oncology Vol. 11 No. 9
of cell-free plasma DNA. Clin Cancer Res. 2014;20:
16981705.
3. Maheswaran S, Sequist LV, Nagrath S, et al. Detection of
mutations in EGFR in circulating lung-cancer cells. N Engl
J Med. 2008;359:366377.
4. Tsao SC-H, Weiss J, Hudson C, et al. Monitoring response
to therapy in melanoma by quantifying circulating tumour
DNA with droplet digital PCR for BRAF and NRAS muta-
tions. Sci Rep. 2015;5:11198.
5. Shibata T, Kokubu A, Miyamoto M, Sasajima Y, Yamazaki N.
Mutant IDH1 confers an in vivo growth in a melanoma
cell line with BRAF mutation. Am J Pathol. 2011;178:
13951402.