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Detection and Monitoring of the BRAF Mutation in Circulating Tumor Cells and Circulating Tumor DNA in BRAF-Mutated Lung Adenocarcinoma

Journal of Thoracic Oncology, Volume 11, Issue 9, September 2016, Pages e109–e112

Introduction

The B-Raf proto-oncogene, serine/threonine kinase gene (BRAF) V600E mutation occurs in less than 2% of cases of non–small cell lung carcinoma (NSCLC); however, 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) inhibitors.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 and 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).

Table 1

BRAF-Mutated and Wild-Type DNA in Plasma and CTCs during Treatment with BRAF Inhibitors

 

Patient Last Treatment Received (at Time of Blood Collection) CTCs/2 Spots Mutant Copies/mL in CTCs Negative Control (WT Patients) Positive Control (10 ng DNA A375) Mutant Copies/mL in ctDNA WT Copies/mL in ctDNA Mutant/Total cfDNA RECIST Evaluation
1 Dabrafenib
Dabrafenib +trametinib
Dabrafenib +trametinib
1
5
3
0
0
0
0
0
0
145.9
161.7
154.6
600
290
100
590
1250
970
50.4%
18.8%
9.3%
DP
DP
2 Bevacizumab
Vemurafenib
3
3
0
0
0
0
149.6
146.3
27
274
1167
27120
2.3%
1%
DP
3 Pemetrexed
Vemurafenib
Vemurafenib
0
0
0
1.6
0
0
0
0
0
152.8
152.1
146
1.6
0
5.8
350
900
400
0.45%
0%
1.4%
PR
DP
4 Pemetrexed
Vemurafenib
0
0
0
0
0
0
133.2
149.8
0.2
1.4
446
1170
0.04%
0.11%
DP
5 Cisplatin + pemetrexed
Vemurafenib
1
NA
0.04
NA
0
NA
141.1
NA
240
NA
1460
NA
14.1%
NA
DP
6 Pemetrexed
Vemurafenib
0
NA
0.12
NA
0
NA
144.2
NA
272
NA
2815
NA
8.8%
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.

In the first 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 suffered 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 identified, besides the known BRAF mutation, a p.Arg132Cys-IDH1 mutation. For patients 2 through 4 (Fig. 2), for whom first 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).

gr1

Figure 1

Variations in the ratio of B-Raf proto-oncogene, serine/threonine kinase gene (BRAF)-mutated over wild-type (WT) circulating free DNA during treatment with dabrafenib (a B-Raf proto-oncogene, serine/threonine kinase inhibitor [BRAFi]) and trametinib (a mitogen-activated protein kinase inhibitor [MEKi]) in patient 1 and the correlations with a computed tomography scan.

 

gr2

Figure 2

The increase in B-Raf proto-oncogene, serine/threonine kinase gene (BRAF) V600E–mutated circulating tumor-specific 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.

 

Discussion

Rapid, noninvasive, and repeatable access to the molecular profile 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 first 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.4BRAF mutants in cfDNA were often detected in small amounts, but no positive droplets were detected in the WT samples, indicating good specificity (see Table 1).

In patient 1, the decreased BRAF-mutated DNA indicated that the BRAF inhibitor was still active on the BRAF clone. However, the concomitant increase in BRAF WT in cfDNA and tumor progression suggests that this clone was no longer predominant. The isocitrate dehydrogenase (NADP(+)), 1 systolic gene (IDH1) mutation confers in vivo growth of the BRAF-mutated melanoma cell line5 and was probably the mechanism of resistance in this case. No archival tissue was available to confirm that this alteration was not initially present.

Our observations suggest that plasma has better sensitivity compared with CTCs. However, CTCs have several advantages (prognostic value, fluorescence in situ hybridization, immunocytochemistry) but are probably not as effective at detecting and monitoring mutations.

In conclusion, analyses of BRAF mutants using digital droplet PCR on cfDNA is feasible and appears to be more sensitive than analyzing CTCs. This test could be useful when following up BRAF-mutated lung adenocarcinoma.

References

  • 1 D. Planchard, T.M. Kim, J. Mazieres, et al. Dabrafenib in patients with BRAFV600E-positive advanced non-small-cell lung cancer: a single-arm, multicentre, open-label, phase 2 trial. Lancet Oncol. 2016; [e-pub ahead of print]pii: S1470-2045(16)00077-2, accessed April 11, 2016
  • 2 G.R. Oxnard, C.P. Paweletz, Y. Kuang, et al. Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA. Clin Cancer Res. 2014;20:1698-1705 Crossref
  • 3 S. Maheswaran, L.V. Sequist, S. Nagrath, et al. Detection of mutations in EGFR in circulating lung-cancer cells. N Engl J Med. 2008;359:366-377 Crossref
  • 4 S.C.-H. Tsao, J. Weiss, C. Hudson, et al. Monitoring response to therapy in melanoma by quantifying circulating tumour DNA with droplet digital PCR for BRAF and NRAS mutations. Sci Rep. 2015;5:11198
  • 5 T. Shibata, A. Kokubu, M. Miyamoto, Y. Sasajima, N. Yamazaki. Mutant IDH1 confers an in vivo growth in a melanoma cell line with BRAF mutation. Am J Pathol. 2011;178:1395-1402 Crossref

Footnotes

a Thoracic Oncology Department, Larrey Hospital, University Hospital of Toulouse, University of Toulouse III (Paul Sabatier), Toulouse, France

b Inserm, Centre de Recherche en Cancérologie de Toulouse, CRCT UMR-1037, Toulouse, France

c Laboratoire de Biologie Médicale Oncologique, Institut Universitaire du Cancer de Toulouse, France

d Drug Development Department, Gustave Roussy Cancer Campus, Paris-Sud University, Villejuif, France

Corresponding author. Address for correspondence: Julien Mazières, MD, PhD, Thoracic Oncology Unit, Respiratory Disease Department, Hôpital Larrey, CHU Toulouse, Chemin de Pouvourville, 31059 Toulouse Cedex, France.

Disclosure: The authors declare no conflict of interest.

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