Letter to the Editor

Citation: Blood Cancer Journal (2013) 3, e163; doi:10.1038/bcj.2013.60
Published online 22 November 2013

Lack of BRAF V600E mutation in human myeloma cell lines established from myeloma patients with extramedullary disease
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L Lodé1, P Moreau2,3, A Ménard1, C Godon1, C Touzeau2,3,4,5, M Amiot3,4,5, S Le Gouill2,3,4,5, M C Béné1,3 and C Pellat-Deceunynck2,3,4,5

  1. 1Laboratoire d’Hématologie, CHU de Nantes, Nantes, France
  2. 2Service d’Hématologie, CHU Nantes, Nantes, France
  3. 3Université de Nantes, Nantes, France
  4. 4INSERM, UMR892, Nantes, France
  5. 5CNRS, UMR 6299, Nantes, France

Correspondence: C Pellat-Deceunynck, E-mail: catherine.pellat-deceunynck@inserm.fr

After whole genome sequencing of samples from 38 patients with multiple myeloma (MM) had identified one patient with an activating mutation of BRAF (G469A), BRAF mutations have been intensively screened in MM patients.1 Among 161 samples, 3 K601N and 4 V600E (the most common in melanoma) mutations were found in 4% of the patients.1 This low incidence is still consistent with a recent study involving 32 MM samples that reported no BRAF mutation.2 In the more specific field of extramedullary disease (EMD) in MM, Andrulis et al.3 recently reported a significant association with BRAF exon 15 mutations. Notably, the V600E mutation was found in 8.5% of patients with EMD (4 out of 47) versus 1.5% of patients without EMD (3 out of 204). The EMD of the four patients harbouring BRAF V600E mutation (all soft tissue plasmacytomas) was primitive for one patient and secondary after one or several lines of treatments for the three others. This recent work contrasts with an older one in which no BRAF mutation was found in 65 fresh bone marrow samples from 18 patients with PCL and 47 patients with MM at diagnosis.4 The incidence of EMD in MM is rare at diagnosis but extramedullary involvement increases with disease evolution. Spreading of MM cells out of the bone marrow is commonly associated with a poor outcome and resistance to salvage therapies.5 In this context, the recent findings of Andrulis et al. raise the interest of identifying patients with EMD carrying the BRAF V600E mutation, who could benefit from the V600E-mutated BRAF protein targeted therapy, that is, vemurafenib.

The establishment of human myeloma cell lines (HMCLs) remains rare and has mainly been obtained in samples from patients who had massive and/or serous EMD (mostly secondary), whatever the origin of patient’s samples, that is, bone marrow, peripheral blood, pleural effusion or ascites fluid.6, 7 Although these HMCLs mostly derived from end-stage disease, they retained the oncogenic abnormalities found at the time of diagnosis.6, 7 A recent study, which assessed the presence of BRAF mutation in six HMCLs, reported that U266 harboured the K601N mutation, suggesting that BRAF mutation could be frequent in HMCLs.8 We thus screened 33 HMCLs for V600E BRAF mutation by sequencing exon 15 to determine whether vemurafenib could be a common therapeutic approach for patients with massive EMD, especially plasma cell leukaemia. In this collection, 2 HMCLs were derived from ascites fluid, 18 from peripheral blood, 12 from pleural effusion and 1 from subcutaneous sample (Table 1). Unfortunately, none of the HMCLs carried the V600E mutation (Table 1). U266 was retrieved in this screening to harbour the K601N BRAF mutation (66% of mutated allele) and no other BRAF mutation was found in the collection. U266 was subcloned in order to define whether the mutation was present in each cell. As shown in Figure 1a, all clones evaluated (n=17) harboured the mutation with the same proportion of 66%, as found within the parental cell line. Because U266 is known to carry a duplication of the q32q34 region of chromosome 7 where BRAF is located, it is likely that the mutated BRAF allele is duplicated.9 We further determined the sensitivity of U266 and three BRAF wild-type cell lines to vemurafenib. All cell lines displayed a very weak sensitivity with IC50 values higher than 5μM (Figure 1b and Table 1). By contrast, in V600E-mutated melanoma cells, IC50 values were lower than 100nM, whereas V600E unmutated cells (including cells carrying other BRAF mutations) required more than 1μM to display any sensitivity.10 Although the BRAF-mutated HMCL did not carry any RAS mutation, 45% of HMCLs (15 out of 33) harboured a K- or N-RAS activating mutation (Table 1). Our findings show that BRAF mutation, in contrast to that of RAS,1 is a rare event even in massive and/or serous EMD and that targeting BRAF V600E mutation with vemurafenib in MM could unfortunately be of limited value in patients with massive and/or serous EMD, such as pleural effusion or plasma cell leukaemia. Nevertheless, vemurafenib could be of high interest for patients with soft tissue plasmacytomas, in which the BRAF V600E mutation has been found, provided the mutation incidence should be significant in that infrequent MM presentation.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

(a) BRAF exon 15 DNA sequencing was performed in U266 cell line and in 17 clones derived by limiting dilution assay. All sequenced clones harboured the same mutation proportion as illustrated in the figure. (b) Cells (30000 cells per 0.2ml) were seeded for 3 days in the presence of increasing concentrations of vemurafenib (Selleckchem, www.selleckchem.com). Proliferation was determined with the Alamar Blue Assay according to the manufacturer’s instructions (AbD Serotec, Biorad, Marnes La Coquette, France). The data represent the mean±s.e.m. of three independent experiments performed in triplicate wells.

Full figure and legend (85K)


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Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

We thank Fabienne Perrault-Hu, Véronique Chenais and Yevgeniya Zozulya for excellent technical assistance.

Author contributions

LL and CPD designed the study, performed experiments and wrote the paper. PM and MCB participated in writing the paper. AM performed experiments. CG reviewed karyotype. CT, MA and SLG reviewed the manuscript.

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/.