Abstract

Langerhans cell histiocytosis (LCH) is a clinically and histologically heterogeneous disorder. Its classification as either reactive inflammatory or neoplastic has been a matter of debate. However, the recent finding of frequent BRAFV600E mutations in LCH argues for the latter. The exact cell type that harbors the mutation and is responsible for proliferation remains to be identified. We here apply a BRAFV600E mutation-specific antibody to detect the BRAF mutant cells in lesions from 89 patients with LCH. We found BRAFV600E mutations in 34 of 89 (38%) lesions. In lesions with the BRAFV600E mutation, the majority of cells coexpressing S-100 and CD1a harbored mutant BRAFV600E protein. These cells also expressed CD14 and CD36, whereas various fractions exhibited CD207. On the other hand, CD80 and CD86 expression was also present on BRAFV600E-positive cells. Thus, cells of variable maturation, exhibiting an immunohistochemical profile compatible either with myeloid cell or with dedifferentiated Langerhans cell antigens, carry the BRAFV600E mutation. In conclusion, we identify and characterize the neoplastic cells in LCH with BRAFV600E mutations by applying a mutation-specific marker and demonstrate feasibility for routine screening.

Introduction

Langerhans cell histiocytosis (LCH) is a heterogeneous disease affecting patients of different ages, manifesting at different sites and presenting as focal or systemic disease. LCH takes an unpredictable clinical course ranging from spontaneous remission to fatal progression. The lesions in LCH present microscopically as heterogeneous mixtures of cells containing various proportions of lymphocytes, macrophages, giant cells, a prominent population of eosinophilic granulocytes, and histiocytes. Lichtenstein was the first to identify those histiocytes as an abundant and thus defining fraction and chose the term “Histiocytosis X” to point toward the uncertainty about their exact nature.1 

The term “Langerhans cell histiocytosis” was coined on recognition of common morphologic and immunohistochemical features with skin Langerhans cells.2-4  LCH thus was separated from non-Langerhans cell histiocytosis, such as Erdheim-Chester or Rosai-Dorfman disease.

Currently, the diagnostic gold standard for LCH is detection of both CD1a and CD207 in Langerhans cells from LCH lesions (herein referred to as “pathologic LCs”). Expression of S-100 is less specific.2,5-8  The origin of pathologic LCs has not been determined. LCH has been discussed to be either an inflammatory reactive or a neoplastic disease. The mixed composition of the lesions with a prominent lymphocytic and granulocytic component was compatible with inflammation and reactive change. However, 2 independent studies recently detected frequent BRAFV600E mutations by DNA sequencing in LCH, strongly arguing for neoplastic disease.9-12  Because of the heterogeneous mixture of cells in LCH lesions, the exact cell type harboring the BRAF mutation so far has not been established. Mutant BRAF is a constitutive activator of the AKT/ERK kinases pathway driving proliferation and is observed in many types of cancer.13 

DNA sequencing of LCH is difficult because of the mixed composition of the lesions, including neoplastic and reactive cells. We have recently developed a mouse monoclonal antibody (clone VE1) with high specificity for mutant BRAFV600E protein. With this study, we demonstrate the feasibility of VE1 to detect BRAFV600E mutation in LCH. Further, we intended to characterize the neoplastic cells in LCH.

Methods

Immunohistochemistry

Formalin-fixed, paraffin embedded tissue from 89 cases of LCH were obtained from the Institute of Pathology, Heidelberg, the Institute of Pathology at the Medical University of Vienna, the Clinical Institute of Neurology (Neuropathology), Medical University of Vienna, and the Institute of Pathology, Charité-University Medicine, Berlin. Normal skin and tonsil tissues served as controls. Mutant BRAFV600E protein was detected by specific mouse monoclonal antibody VE1.14,15  Immunohistochemistry included cell conditioning with Ventana (Ventana Medical Systems) cell conditioner 1 for 64 minutes, preprimary peroxidase inhibition, incubation with 1:5 diluted VE1 hybridoma supernatant at 37°C for 32 minutes, incubation with OptiView HQ Universal Linker for 12 minutes, incubation with OptiView HRP Multimer for 12 minutes, OptiView Amplification (setting of OptiView Amplifier and OptiView Amplifier Multimer both for 12 minutes), and incubation with hematoxylin and Bluing reagent for 4 minutes each. Although we used hybridoma supernatant, purified VE1 is now commercially available (Spring Bioscience). Meanwhile, studies applying different detection systems have also been published,14-16  one discussing advantages of both methods under different conditions in detail.15 

Immunohistochemistry for CD207 (1:25; Atlas Antibodies), CD1a (1:25; Thermo Scientific), Ki-67 (1:100; Medac), p53 (1:50; Novocastra, distributed by Leica), S-100 (1:100; Dako Denmark), and pERK (1:100; Cell Signaling), CD34 (1:2; Thermo Scientific) included pretreatment with either Ventana cell conditioner 1 (CD207, CD1a, Ki-67, p53, CD34) or protease for 4 minutes (pERK) and antibody incubation for 32 minutes at 37°C. Incubation was followed by Ventana standard signal amplification, UltraWash, counterstaining with 1 drop of hematoxylin for 4 minutes, and 1 drop of bluing reagent for 4 minutes. For visualization, ultraViewUniversal DAB or FastRed (S-100) Detection Kit (Ventana) was used. Images were acquired on an Axioplan2 microscope (Carl Zeiss) equipped with an AxioCamHR camera and AxioVision Version 4.8 (both Carl Zeiss) software. Magnifications are given in figure legends.

For immunofluorescence, slides were deparaffinized according to standard protocols and underwent antigen retrieval for 30 minutes in cell conditioner 1 (Ventana) in a steam cooker. Primary antibodies VE1, CD33 (1:50; Santa Cruz Biotechnology), CD36 (1:50), CD80 (1:50), CD86 (1:50), CD14 (1:100, all by Abcam), CD3 (1:50; Dako), and CD207 (1:50) were incubated overnight at 4°C. VE1 was detected applying the Tyramide 488 Alexa TSA kit (Invitrogen) according to the manufacturer's instructions. Other antibodies were detected by incubation with AlexaFluor-568 nm (1:500; Invitrogen) for 30 minutes at room temperature. Images were obtained using an Olympus BX50 fluorescence microscope with ×100 objective lens (Olympus), equipped with an AxioCamMR3 camera and AxioVision Version 4.8 software (both Carl Zeiss).

PCR amplification and direct sequencing

DNA was extracted from 46 samples applying the Invisorb DNA extraction kit (Stratek) according to the manufacturer's instructions. Then, a fragment spanning the catalytic domain of BRAF including codon 600 was amplified and sequenced as previously described.17  In brief, PCR was performed in a total volume of 15 μL, containing 20 ng of DNA and 2 times PCR Master Mix (Promega) with initial denaturation at 95°C for 60 seconds, followed by 35 cycles with denaturation at 95°C for 30 seconds, annealing at 56°C for 40 seconds, and extension at 72°C for 50 seconds. A total of 2 μL of the amplification product was submitted to sequencing using the BigDye Terminator Version 3.1 Sequencing Kit (Applied Biosystems). Twenty-five cycles were performed using 12 ng of the sense primer, with denaturation at 95°C for 30 seconds, annealing at 56°C for 15 seconds, and extension at 60°C for 240 seconds. Sequences were determined performing bidirectional direct sequencing on a semiautomated sequencer (ABI 3100 Genetic Analyzer; Applied Biosystems) and the Sequence Pilot Version 3.1 (JSI-Medisys) software.

Results

Mutant protein and BRAFV600 mutations in 89 LHC lesions

The presence or absence of the BRAFV600E mutation was determined in all 89 lesions by VE1 immunohistochemistry. Mutant protein was detected in 34 of 89 (38%) cases. DNA for direct sequencing of the BRAFV600 region was available from 46 patients. A BRAFV600E mutation was detected in 18 of 46 (39%) lesions, a V600K mutation in 1 of 46 lesions (2%), and a wild-type status in 27 of 46 (59%) lesions. In 1 lesion with DNA available, the BRAFV600E alteration was detected by immunohistochemistry only and not by direct sequencing. The single lesion with the BRAFV600K mutation was not detected by antibody VE1. Thus, sensitivity for detection of the V600 mutations was 95% for immunohistochemistry and 95% for direct sequencing. Concordant results of immunohistochemistry and direct sequencing were seen in 96% of the cases. The tissue set assessed by immunohistochemistry only yielded comparable results, with 15 of 43 (35%) mutant cases (Table 1). Because previous reports observed a germline mutation in the BRAF gene,12  we were alert to this possibility. However, no case was observed where VE1 positivity was found in adjacent tissue. Age distribution of patients with mutant versus wild-type LCH lesions revealed no significant difference (P > .65, Student t test). Patient characteristics and BRAF V600 status are compiled in Table 1. As far as retrievable from medical records, 4 patients (cases 3, 16, 17, and 73) had multifocal lesions. This did not allow for statistical analysis of multifocal versus focal disease.

Table 1

LCH lesions assessed for BRAF mutations both immunohistochemically and by direct sequencing (Set 1) and samples with tissue amounts only sufficient for IHC (Set 2)

Set 1
 
Sequencing Set 2
 
ID no. Age, y Origin of tissue examined IHC ID no. Age, y Origin of tissue examined IHC 
39 Upper GI mucosa + V600E 47 NA + 
Bone + V600E 48 22 Bone + 
63 Spleen + V600E 49 24 Connective tissue + 
23 Bone + V600E 50 21 Bone + 
Bone + V600E 51 47 Bone + 
Bone + V600E 52 27 Bone + 
13 Bone + V600E 53 16 Bone + 
Bone + V600E 54 12 Bone + 
27 Bone + V600E 55 Bone + 
10 10 Bone + V600E 56 NA + 
11 12 Bone + V600E 57 NA + 
12 13 Bone + V600E 58 47 Bone + 
13 13 Bone + V600E 59 19 Skin + 
14 21 Bone + V600E 60 Spleen + 
15 41 Bone + V600E 61 80 Bone + 
16 NA Uterus + V600E 62 14 Bone − 
17 63 Upper GI mucosa + V600E 63 Bone − 
18 11 Bone − V600K 64 12 Upper GI mucosa − 
19 74 Lymph node + V600E 65 Bone − 
20 38 Bone + WT 66 Bone − 
21 26 Bone − WT 67 14 Bone − 
22 38 Bone − WT 68 19 Bone − 
23 26 CNS − WT 69 Bone − 
24 Skin − WT 70 13 NA − 
25 56 Upper GI mucosa − WT 71 NA − 
26 22 Bone − WT 72 17 NA − 
27 34 Bone − WT 73 31 CNS − 
28 21 Bone − WT 74 12 NA − 
29 34 Thyroid gland − WT 75 NA − 
30 Bone − WT 76 14 NA − 
31 Muscle − WT 77 12 NA − 
32 25 Bone − WT 78 NA − 
33 22 Bone − WT 79 16 Bone − 
34 79 Bone − WT 80 NA − 
35 NA Skin − WT 81 34 Bone − 
36 35 Bone − WT 82 20 Lung − 
37 45 Skin − WT 83 Skin − 
38 27 Skin − WT 84 Bone − 
39 43 Bone − WT 85 20 NA − 
40 38 Lymph node − WT 86 16 NA − 
41 23 Bone − WT 87 17 Bone − 
42 Bone − WT 88 Bone − 
43 Bone − WT 89 58 Bone − 
44 10 Bone − WT     
45 Lymph node − WT     
46 10 Bone − WT     
Set 1
 
Sequencing Set 2
 
ID no. Age, y Origin of tissue examined IHC ID no. Age, y Origin of tissue examined IHC 
39 Upper GI mucosa + V600E 47 NA + 
Bone + V600E 48 22 Bone + 
63 Spleen + V600E 49 24 Connective tissue + 
23 Bone + V600E 50 21 Bone + 
Bone + V600E 51 47 Bone + 
Bone + V600E 52 27 Bone + 
13 Bone + V600E 53 16 Bone + 
Bone + V600E 54 12 Bone + 
27 Bone + V600E 55 Bone + 
10 10 Bone + V600E 56 NA + 
11 12 Bone + V600E 57 NA + 
12 13 Bone + V600E 58 47 Bone + 
13 13 Bone + V600E 59 19 Skin + 
14 21 Bone + V600E 60 Spleen + 
15 41 Bone + V600E 61 80 Bone + 
16 NA Uterus + V600E 62 14 Bone − 
17 63 Upper GI mucosa + V600E 63 Bone − 
18 11 Bone − V600K 64 12 Upper GI mucosa − 
19 74 Lymph node + V600E 65 Bone − 
20 38 Bone + WT 66 Bone − 
21 26 Bone − WT 67 14 Bone − 
22 38 Bone − WT 68 19 Bone − 
23 26 CNS − WT 69 Bone − 
24 Skin − WT 70 13 NA − 
25 56 Upper GI mucosa − WT 71 NA − 
26 22 Bone − WT 72 17 NA − 
27 34 Bone − WT 73 31 CNS − 
28 21 Bone − WT 74 12 NA − 
29 34 Thyroid gland − WT 75 NA − 
30 Bone − WT 76 14 NA − 
31 Muscle − WT 77 12 NA − 
32 25 Bone − WT 78 NA − 
33 22 Bone − WT 79 16 Bone − 
34 79 Bone − WT 80 NA − 
35 NA Skin − WT 81 34 Bone − 
36 35 Bone − WT 82 20 Lung − 
37 45 Skin − WT 83 Skin − 
38 27 Skin − WT 84 Bone − 
39 43 Bone − WT 85 20 NA − 
40 38 Lymph node − WT 86 16 NA − 
41 23 Bone − WT 87 17 Bone − 
42 Bone − WT 88 Bone − 
43 Bone − WT 89 58 Bone − 
44 10 Bone − WT     
45 Lymph node − WT     
46 10 Bone − WT     

IHC indicates immunohistochemistry; Sequencing, V600E/K mutation in BRAF and respective amino acid exchange; GI, gastrointestinal; WT, wild-type; and NA, data not available.

Cell characterization by immunohistochemistry and immunofluorescence

We next assessed the phenotype of the BRAFV600E mutant cells in 3 different LCH lesions (cases 9, 19, and 47). Immunohistochemistry of consecutive sections demonstrated a wide overlap of BRAFV600E mutant cells (Figure 1A-B) with classic antigens expressed by Langerhans cells, such as S-100 (Figure 1C-D), CD207 (Figure 1E-F), and CD1a (Figure 1G-H). For confirmation, CD207-expressing cells from a representative lesion (case 9) with VE1 positivity were sampled by laser capture microdissection. Consecutive sequencing revealed a BRAFV600E mutation in this fraction (data not shown). Of note, in some cases, the number of CD207-expressing cells (Figure 1H) was lower than the number of VE1-positive cells (Figure 1B). This heterogeneity could also be verified by immunofluorescence (Figure 2) performed on 3 independent LCH lesions (cases 1, 9, and 19). Although the majority of cells expressed both mutant BRAFV600E protein and CD207 (Figure 2A-I), single cells only yielded a signal for BRAFV600E (Figure 2D-E). All BRAFV600E-positive cells expressed the antigen CD36, which is frequently found in myeloid dendritic cells18-21  (Figure 3A-C) and the monocyte marker CD14 (Figure 3D-F) but lacked expression of the T-cell lineage marker CD3 (Figure 3G-I). CD33 typically strongly expressed in mature Langerhans cells within the skin (Figure 3L)21,22  was rarely found in LCH lesions (Figure 3J-K). Moreover, BRAFV600E-positive cells also expressed CD80 (Figure 3L-O) and some also CD86 (supplemental Figure 1A-C, see the Supplemental Materials link at the top of the article), both up-regulated during LC maturation,6,22-24  but rarely the hematopoietic progenitor antigen CD34,22  which was mainly limited to vessels (supplemental Figure 1D-E).

Figure 1

Immunohistochemistry for BRAFV600E, CD1a, CD207, and S-100 in serial sections of 2 representative cases. One reveals high overlap between mutant and CD207-positive cells (case 9, left panels), and the other reveals a minority of mutant cells expressing CD207 (case 19, right panels, ×10 lens).

Figure 1

Immunohistochemistry for BRAFV600E, CD1a, CD207, and S-100 in serial sections of 2 representative cases. One reveals high overlap between mutant and CD207-positive cells (case 9, left panels), and the other reveals a minority of mutant cells expressing CD207 (case 19, right panels, ×10 lens).

Figure 2

Representative immunofluorescent labeling. (A,D,G) CD207. (B,E,H) BRAFV600E. The left and central panels underline the wide overlap of mutant protein and CD207-expressing cells. (C,F,I) Overlay. Arrow indicates mutant BRAFV600E protein in a cell without signal for CD207.

Figure 2

Representative immunofluorescent labeling. (A,D,G) CD207. (B,E,H) BRAFV600E. The left and central panels underline the wide overlap of mutant protein and CD207-expressing cells. (C,F,I) Overlay. Arrow indicates mutant BRAFV600E protein in a cell without signal for CD207.

Figure 3

Representative immunofluorescence. BRAFV600E combined with antibodies against CD36 (A-C), CD14 (D-F), CD3 (G-I), CD33 (J-K; skin sample stained for CD33 as positive control, L), and CD80 (M-O).

Figure 3

Representative immunofluorescence. BRAFV600E combined with antibodies against CD36 (A-C), CD14 (D-F), CD3 (G-I), CD33 (J-K; skin sample stained for CD33 as positive control, L), and CD80 (M-O).

Distribution of BRAFV600E-positive cells and activation of pERK

In the majority of the 34 cases with BRAFV600E, mutant protein was primarily detected in granuloma-like structures; however, single dispersed cells were also observed (Figure 4A). The ratio of BRAFV600E-positive cells to cells not harboring mutant protein varied from 3% to 90%. Accumulation of BRAFV600E-positive cells was observed in the regions with highest Ki-67 index and mitotic figures (Figure 4A). As determined on consecutive sections stained for S-100, CD1a, and Ki-67 (illustrated in supplemental Figure 1F-H), there was, however, no difference in proliferation rates of the respective cells in lesions with and without BRAFV600E mutations (Figure 4B, P > .61, Student t test). Similarly, the activation of the RAS/RAF/MEK/ERK signal cascade, also determined with regard to areas of CD1a+ and S-100+ cells, did not differ between lesions with and without BRAFV600E mutations (Figure 4E; P > .24, Student t test). This was assessed in at least 8 LCH lesions with and in 8 LCH lesions without BRAFV600E mutation. Because TP53 mutations and subsequent up-regulation of mutant p53 protein have also been discussed as a pathomechanism underlying LCH,25,26  we assessed p53 expression in the lesions by immunohistochemistry. Strong expression of p53 was seen in LCH independent of BRAFV600E mutational status (Figure 4F; P > .19, Student t test). However, p53 was absent in Langerhans cells from the skin (data not shown).

Figure 4

BRAFV600E expression. Areas with increased mitotic activity (A, inset), percentage of cells stained for Ki-67 in mutant and wild-type cases (B), percentage of cells stained for pERK in mutant and wild-type cases (C), representative immunohistochemistry for pERK in a mutant (D, case 11) and wild-type case (E, case 26, ×10 lens), and percentage of cells strongly stained for p53 (F).

Figure 4

BRAFV600E expression. Areas with increased mitotic activity (A, inset), percentage of cells stained for Ki-67 in mutant and wild-type cases (B), percentage of cells stained for pERK in mutant and wild-type cases (C), representative immunohistochemistry for pERK in a mutant (D, case 11) and wild-type case (E, case 26, ×10 lens), and percentage of cells strongly stained for p53 (F).

Discussion

Analysis of LCH is particularly challenging because it is composed of different cell types that are heterogeneously distributed within the lesions. Precise evaluation, therefore, is greatly assisted by tools successfully identifying individual cell populations. Several studies dissected LCH lesions with specific cellular lineage or differentiation markers, such as CD1a, CD207, CD34, CD3, and others.2,6,27,28  However, none of these markers could unequivocally substantiate the neoplastic cell component in these heterogeneous lesions. Furthermore, the classification of LCH as either reactive or neoplastic disease had not been resolved until the recent detection of activating BRAF mutations in approximately half of LCH lesions. This observation favors the classification of LCH as a neoplastic disease.9,11,12,29  So far, detection of BRAF mutations had to rely on DNA analysis of the heterogeneous cell populations in LCH. In the present study, we characterize the neoplastic component of LCH with BRAFV600E mutations using the mutation-specific monoclonal antibody VE1.

Mutant BRAF protein was detected by VE1 in 34 of 89 (38%) LCH lesions. This is lower than the frequencies of BRAF mutations in LCH previously published9,12  and is possibly the result of less multifocal cases being included which tended to have higher mutation rates. We could perform both VE1 immunohistochemistry and direct sequencing in 46 of the 89 LCH lesions. Of these 46 lesions, 20 were identified harboring a BRAF mutation. In 18 lesions, the mutation was detected by both methods. One V600K mutation was missed by VE1 not able to detect this alteration, and one V600E mutation was missed by sequencing because of the low number of tumor cells (20, Figure 5). Sanger sequencing requires ∼ 10% mutant alleles for detection in our experience and according to reports from other laboratories.30,31  Indeed, 1 lesion (case 19) was initially also missed by direct sequencing but could be identified on immunohistochemistry-guided selection of tissue for DNA extraction. Thus, there was no significant difference in the sensitivity for detection of BRAF mutations between direct sequencing and immunohistochemistry. This receives further support by the observation of the majority of BRAF mutations in LCH being of the V600E type with only a single mutation detected in position 599.9,12 

Figure 5

Case with binding of mutation-specific antibody but no mutation detectable by sequencing (case 20, ×4 lens).

Figure 5

Case with binding of mutation-specific antibody but no mutation detectable by sequencing (case 20, ×4 lens).

Previous analyses of cells from LCH lesions sorted for CD207 underlined these cells as the disease-determining population.2  Our data clearly confirm this concept, demonstrating coexpression of mutant BRAF protein and CD207 (Figure 2), thus proving the CD207 cells being clonal and neoplastic. However, our data also extend previous observations by supporting the existence and illustrating the extent of neoplastic cells not expressing CD207. First, we can demonstrate a significant difference in the abundance of VE1 and CD207-positive cells in selected cases (Figure 1B,H). Second, applying double-immunofluorescence analysis, we could detect cells positive for VE1 but not for CD207 (Figure 2D-F). Thus, BRAFV600E mutations appear not to be exclusively restricted to pathologic LCs but may rather occur in a population of cells of different maturation, with pathologic LCs constituting the most advanced differentiation status.

Two origins for pathologic LCs have been proposed: one is development from immature myeloid cells in the bone marrow; the other is development from peripheral LCs.2,9,11,23,32  Our findings are compatible with development from immature myeloid cells because of detection of BRAFV600E mutations in both CD207-positive pathologic LCs and in cells expressing CD14 and CD36 but not CD207. The initial BRAF mutation may occur in a less mature myeloid cell with most, but not all, of them differentiating to pathologic LCs. However, these results do not exclude the possibility that mature LCs or their precursors alter their expression profile after acquiring a BRAF mutation or another respective alteration. Indeed, the finding of CD80/CD86 expression on pathologic LCs is well compatible with that hypothesis and in line with previous investigations.6,24  Moreover, in 2 patients (cases 2 and 17) available for analysis, we did not detect BRAFV600E in bone marrow, although the mutation was present in their LCH lesions. This supports previous observations of not detecting BRAFV600E in peripheral blood of LCH patients with mutant lesions.12  Thus, although we do succeed in further characterizing the pathologic cells in LCH, we cannot resolve the debate on their origin.

Investigations on the existence of BRAF mutations in non-LCH histiocytosis with different neoplastic cells might provide further insight into these mechanisms. Similar to LCH, there have been reports on Erdheim-Chester disease establishing clonality by X-chromosome inactivation analysis33  and cytogenetics,34  making this disease also a candidate for neoplastic rather than reactive inflammatory disease. Very recent findings demonstrate BRAFV600E mutations in Erdheim-Chester disease.35 

BRAFV600E mutations constitutively activate the RAS/RAF/MEK/ERK signal transduction cascade. Consequently, we detected phosphorylated ERK in these lesions. However, phosphorylated ERK was also present in comparable amounts in LCH lesions without BRAF mutations (Figure 4). Our observation confirms previous findings9  and suggests activation of the MAPK/ERK/AKT cascade and perturbation of its regulation by mechanisms other than BRAF point mutations. Similarly, the proliferation activity did not differ in LCH lesions with and without BRAF mutations.

In conclusion, we demonstrate the ability of monoclonal antibody VE1 to detect BRAF mutation in LCH with high sensitivity and the potential of VE1 to assist in determination of the origin of pathologic LCs in these lesions. Immunohistochemical detection of BRAF mutations may greatly assist the diagnosis of LCH, especially in cases with a low proportion of pathologic LCs. Furthermore, it may help direct BRAF mutation-targeting therapy to these patients. The BRAF inhibitors vemurafenib and dabrafenib have shown evidence of therapeutic activity in several BRAFV600E-mutated cancer types, including metastatic melanoma, refractory hairy cell leukemia, and others,16,36,37  and might also be a treatment option for patients with LCH harboring BRAFV600E mutations. It remains to be seen by analysis of large patient cohorts whether the BRAF mutation characterizes a distinct subgroup among LCH.

This article contains a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Acknowledgments

The authors thank Kerstin Lindenberg and Jutta Scheuerer for skillful technical assistance.

Authorship

Contribution: F.S. and A.v.D. designed the study, analyzed data, and wrote the manuscript; F.S., J.M., A.H., and F.L. performed experiments; D.C., M.P., A.S., A.-S.B., M.S., A.K., I.A., L.M., and G.M. provided tissue samples and patient information and analyzed the data; and all authors were involved in manuscript preparation.

Conflict-of-interest disclosure: A.v.D. and D.C. declare shared inventorship of BRAF antibody clone VE1. A patent for diagnostic application of VE1 has been applied for. All terms are being managed by the German Cancer Research Center in accordance with its conflict-of-interest policies. The remaining authors declare no competing financial interests.

Correspondence: Andreas von Deimling, Department of Neuropathology, Institute of Pathology, Ruprecht-Karls-Universität Heidelberg, D-69120 Heidelberg, Germany; e-mail: andreas.vondeimling@med.uni-heidelberg.de.

References

References
1
Lichtenstein
 
L
Histiocytosis X: integration of eosinophilic granuloma of bone, Letterer-Siwe disease, and Schuller-Christian disease as related manifestations of a single nosologic entity.
AMA Arch Pathol
1953
, vol. 
56
 
1
(pg. 
84
-
102
)
2
Allen
 
CE
Li
 
L
Peters
 
TL
, et al. 
Cell-specific gene expression in Langerhans cell histiocytosis lesions reveals a distinct profile compared with epidermal Langerhans cells.
J Immunol
2010
, vol. 
184
 
8
(pg. 
4557
-
4567
)
3
Valladeau
 
J
Ravel
 
O
Dezutter-Dambuyant
 
C
, et al. 
Langerin, a novel C-type lectin specific to Langerhans cells, is an endocytic receptor that induces the formation of Birbeck granules.
Immunity
2000
, vol. 
12
 
1
(pg. 
71
-
81
)
4
Mierau
 
GW
Favara
 
BE
Brenman
 
JM
Electron microscopy in histiocytosis X.
Ultrastruct Pathol
1982
, vol. 
3
 
2
(pg. 
137
-
142
)
5
Jaffe
 
R
The Diagnostic Histopathology of Langerhans Cell Histiocytosis
2005
Cambridge, United Kingdom
Cambridge University Press
6
Geissmann
 
F
Lepelletier
 
Y
Fraitag
 
S
, et al. 
Differentiation of Langerhans cells in Langerhans cell histiocytosis.
Blood
2001
, vol. 
97
 
5
(pg. 
1241
-
1248
)
7
Valladeau
 
J
Duvert-Frances
 
V
Pin
 
JJ
, et al. 
The monoclonal antibody DCGM4 recognizes Langerin, a protein specific of Langerhans cells, and is rapidly internalized from the cell surface.
Eur J Immunol
1999
, vol. 
29
 
9
(pg. 
2695
-
2704
)
8
Emile
 
JF
Wechsler
 
J
Brousse
 
N
, et al. 
Langerhans' cell histiocytosis: definitive diagnosis with the use of monoclonal antibody O10 on routinely paraffin-embedded samples.
Am J Surg Pathol
1995
, vol. 
19
 
6
(pg. 
636
-
641
)
9
Badalian-Very
 
G
Vergilio
 
JA
Degar
 
BA
, et al. 
Recurrent BRAF mutations in Langerhans cell histiocytosis.
Blood
2010
, vol. 
116
 
11
(pg. 
1919
-
1923
)
10
Nichols
 
KE
Arceci
 
RJ
BRAF, a piece of the LCH puzzle.
Blood
2010
, vol. 
116
 
11
(pg. 
1825
-
1827
)
11
Badalian-Very
 
G
Vergilio
 
JA
Degar
 
BA
Rodriguez-Galindo
 
C
Rollins
 
BJ
Recent advances in the understanding of Langerhans cell histiocytosis.
Br J Haematol
2012
, vol. 
156
 
2
(pg. 
163
-
172
)
12
Satoh
 
T
Smith
 
A
Sarde
 
A
, et al. 
B-RAF mutant alleles associated with Langerhans cell histiocytosis, a granulomatous pediatric disease.
PLoS One
2012
, vol. 
7
 
4
pg. 
e33891
 
13
Flaherty
 
KT
Narrative review: BRAF opens the door for therapeutic advances in melanoma.
Ann Intern Med
2010
, vol. 
153
 
9
(pg. 
587
-
591
)
14
Capper
 
D
Preusser
 
M
Habel
 
A
, et al. 
Assessment of BRAF V600E mutation status by immunohistochemistry with a mutation-specific monoclonal antibody.
Acta Neuropathol
2011
, vol. 
122
 
1
(pg. 
11
-
19
)
15
Andrulis
 
M
Penzel
 
R
Weichert
 
W
von Deimling
 
A
Capper
 
D
Application of a BRAF V600E mutation-specific antibody for the diagnosis of hairy cell leukemia [published online ahead of print April 22, 2012].
Am J Surg Pathol
 
16
Dietrich
 
S
Glimm
 
H
Andrulis
 
M
von Kalle
 
C
Ho
 
AD
Zenz
 
T
BRAF inhibition in refractory hairy-cell leukemia.
N Engl J Med
2012
, vol. 
366
 
21
(pg. 
2038
-
2040
)
17
Schindler
 
G
Capper
 
D
Meyer
 
J
, et al. 
Analysis of BRAF V600E mutation in 1,320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma, ganglioglioma and extra-cerebellar pilocytic astrocytoma.
Acta Neuropathol
2011
, vol. 
121
 
3
(pg. 
397
-
405
)
18
Albert
 
ML
Pearce
 
SF
Francisco
 
LM
, et al. 
Immature dendritic cells phagocytose apoptotic cells via alphavbeta5 and CD36, and cross-present antigens to cytotoxic T lymphocytes.
J Exp Med
1998
, vol. 
188
 
7
(pg. 
1359
-
1368
)
19
Taylor
 
RS
Baadsgaard
 
O
Hammerberg
 
C
Cooper
 
KD
Hyperstimulatory CD1a+CD1b+CD36+ Langerhans cells are responsible for increased autologous T lymphocyte reactivity to lesional epidermal cells of patients with atopic dermatitis.
J Immunol
1991
, vol. 
147
 
11
(pg. 
3794
-
3802
)
20
Huh
 
HY
Pearce
 
SF
Yesner
 
LM
Schindler
 
JL
Silverstein
 
RL
Regulated expression of CD36 during monocyte-to-macrophage differentiation: potential role of CD36 in foam cell formation.
Blood
1996
, vol. 
87
 
5
(pg. 
2020
-
2028
)
21
Santegoets
 
SJ
Gibbs
 
S
Kroeze
 
K
, et al. 
Transcriptional profiling of human skin-resident Langerhans cells and CD1a+ dermal dendritic cells: differential activation states suggest distinct functions.
J Leukoc Biol
2008
, vol. 
84
 
1
(pg. 
143
-
151
)
22
Mutyambizi
 
K
Berger
 
CL
Edelson
 
RL
The balance between immunity and tolerance: the role of Langerhans cells.
Cell Mol Life Sci
2009
, vol. 
66
 
5
(pg. 
831
-
840
)
23
Emile
 
JF
Fraitag
 
S
Leborgne
 
M
de Prost
 
Y
Brousse
 
N
Langerhans' cell histiocytosis cells are activated Langerhans' cells.
J Pathol
1994
, vol. 
174
 
2
(pg. 
71
-
76
)
24
Tazi
 
A
Moreau
 
J
Bergeron
 
A
Dominique
 
S
Hance
 
AJ
Soler
 
P
Evidence that Langerhans cells in adult pulmonary Langerhans cell histiocytosis are mature dendritic cells: importance of the cytokine microenvironment.
J Immunol
1999
, vol. 
163
 
6
(pg. 
3511
-
3515
)
25
Bank
 
MI
Rengtved
 
P
Carstensen
 
H
Petersen
 
BL
p53 expression in biopsies from children with Langerhans cell histiocytosis.
J Pediatr Hematol Oncol
2002
, vol. 
24
 
9
(pg. 
733
-
736
)
26
Weintraub
 
M
Bhatia
 
KG
Chandra
 
RS
Magrath
 
IT
Ladisch
 
S
p53 expression in Langerhans cell histiocytosis.
J Pediatr Hematol Oncol
1998
, vol. 
20
 
1
(pg. 
12
-
17
)
27
Rolland
 
A
Guyon
 
L
Gill
 
M
, et al. 
Increased blood myeloid dendritic cells and dendritic cell-poietins in Langerhans cell histiocytosis.
J Immunol
2005
, vol. 
174
 
5
(pg. 
3067
-
3071
)
28
Herbst
 
B
Kohler
 
G
Mackensen
 
A
Veelken
 
H
Mertelsmann
 
R
Lindemann
 
A
CD34+ peripheral blood progenitor cell and monocyte derived dendritic cells: a comparative analysis.
Br J Haematol
1997
, vol. 
99
 
3
(pg. 
490
-
499
)
29
Wilejto
 
M
Abla
 
O
Langerhans cell histiocytosis and Erdheim-Chester disease.
Curr Opin Rheumatol
2012
, vol. 
24
 
1
(pg. 
90
-
96
)
30
Tsiatis
 
AC
Norris-Kirby
 
A
Rich
 
RG
, et al. 
Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications.
J Mol Diagn
2010
, vol. 
12
 
4
(pg. 
425
-
432
)
31
Milbury
 
CA
Li
 
J
Makrigiorgos
 
GM
COLD-PCR-enhanced high-resolution melting enables rapid and selective identification of low-level unknown mutations.
Clin Chem
2009
, vol. 
55
 
12
(pg. 
2130
-
2143
)
32
Geissmann
 
F
Manz
 
MG
Jung
 
S
Sieweke
 
MH
Merad
 
M
Ley
 
K
Development of monocytes, macrophages, and dendritic cells.
Science
2010
, vol. 
327
 
5966
(pg. 
656
-
661
)
33
Chetritt
 
J
Paradis
 
V
Dargere
 
D
, et al. 
Chester-Erdheim disease: a neoplastic disorder.
Hum Pathol
1999
, vol. 
30
 
9
(pg. 
1093
-
1096
)
34
Vencio
 
EF
Jenkins
 
RB
Schiller
 
JL
, et al. 
Clonal cytogenetic abnormalities in Erdheim-Chester disease.
Am J Surg Pathol
2007
, vol. 
31
 
2
(pg. 
319
-
321
)
35
Haroche
 
J
Charlotte
 
F
Arnaud
 
L
, et al. 
High prevalence of BRAF V600E mutations in Erdheim-Chester Disease but not in other non-Langerhans cell histiocytoses [published online ahead of print August 9, 2012].
Blood
 
36
Chapman
 
PB
Hauschild
 
A
Robert
 
C
, et al. 
Improved survival with vemurafenib in melanoma with BRAF V600E mutation.
N Engl J Med
2011
, vol. 
364
 
26
(pg. 
2507
-
2516
)
37
Falchook
 
GS
Long
 
GV
Kurzrock
 
R
, et al. 
Dabrafenib in patients with melanoma, untreated brain metastases, and other solid tumours: a phase 1 dose-escalation trial.
Lancet
2012
, vol. 
379
 
9829
(pg. 
1893
-
1901
)