Recurrent NRAS mutations in pulmonary Langerhans cell histiocytosis

  1. Samia Mourah1,
  2. Alexandre How-Kit2,
  3. Véronique Meignin3,
  4. Dominique Gossot4,
  5. Gwenaël Lorillon5,
  6. Emmanuelle Bugnet5,
  7. Florence Mauger6,
  8. Celeste Lebbe7,
  9. Sylvie Chevret8,9,
  10. Jörg Tost6 and
  11. Abdellatif Tazi5,9⇑
  1. 1Assistance Publique – Hôpitaux de Paris, Laboratoire de Pharmacologie Biologique, Hôpital Saint-Louis; Université Paris-Diderot, Sorbonne Paris Cité; INSERM U976, Paris, France
  2. 2Laboratoire de Génomique fonctionnelle, Fondation Jean Dausset – CEPH, Paris, France
  3. 3Assistance Publique – Hôpitaux de Paris, Service de Pathologie, Hôpital Saint-Louis; INSERM UMR_S1165, Paris, France
  4. 4Département Thoracique, Institut Mutualiste Montsouris, Paris, France
  5. 5Assistance Publique – Hôpitaux de Paris, Centre National de Référence de l'Histiocytose Langerhansienne, Service de Pneumologie, Hôpital Saint-Louis, Paris, France
  6. 6Laboratoire Epigénétique et Environnement, Centre National de Génotypage, CEA-Institut de Génomique, Evry, France
  7. 7Assistance Publique – Hôpitaux de Paris, Département de Dermatologie, Hôpital Saint-Louis; Université Paris-Diderot, Sorbonne Paris Cité; INSERM U976, Paris, France
  8. 8Assistance Publique – Hôpitaux de Paris; Service de Biostatistique et Information Médicale, Hôpital Saint-Louis, Paris, France
  9. 9Université Paris-Diderot, Sorbonne Paris Cité; INSERM UMR 1153 CRESS, Equipe de Recherche en Biostatistiques et Epidémiologie Clinique, Paris, France
  1. Abdellatif Tazi, Service de Pneumologie, Hôpital Saint-Louis, 1 Avenue Claude Vellefaux, 75475, Paris cedex 10, France. E-mail: abdellatif.tazi{at}sls.aphp.fr

Abstract

The mitogen-activated protein kinase (MAPK) pathway is constantly activated in Langerhans cell histiocytosis (LCH). Mutations of the downstream kinases BRAF and MAP2K1 mediate this activation in a subset of LCH lesions. In this study, we attempted to identify other mutations which may explain the MAPK activation in nonmutated BRAF and MAP2K1 LCH lesions.

We analysed 26 pulmonary and 37 nonpulmonary LCH lesions for the presence of BRAF, MAP2K1, NRAS and KRAS mutations. Grossly normal lung tissue from 10 smoker patients was used as control. Patient spontaneous outcomes were concurrently assessed.

BRAFV600E mutations were observed in 50% and 38% of the pulmonary and nonpulmonary LCH lesions, respectively. 40% of pulmonary LCH lesions harboured NRASQ61K/R mutations, whereas no NRAS mutations were identified in nonpulmonary LCH biopsies or in lung tissue control. In seven out of 11 NRASQ61K/R-mutated pulmonary LCH lesions, BRAFV600E mutations were also present. Separately genotyping each CD1a-positive area from the same pulmonary LCH lesion demonstrated that these concurrent BRAF and NRAS mutations were carried by different cell clones. NRASQ61K/R mutations activated both the MAPK and AKT (protein kinase B) pathways. In the univariate analysis, the presence of concurrent BRAFV600E and NRASQ61K/R mutations was significantly associated with patient outcome.

These findings highlight the importance of NRAS genotyping of pulmonary LCH lesions because the use of BRAF inhibitors in this context may lead to paradoxical disease progression. These patients might benefit from MAPK kinase inhibitor-based treatments.

Abstract

Pulmonary Langerhans cell histiocytosis genetic landscape includes recurrent activating NRAS Q61 mutations http://ow.ly/YgsSm

Introduction

Langerhans cell histiocytosis (LCH) is a rare disorder of unknown origin that encompasses a large clinical spectrum [1]. Lung involvement is frequently observed in young adult smokers and exhibits variable outcomes [2–4]. Alterations in the mitogen-activated protein kinase (MAPK) pathway are involved in the pathogenesis of LCH [5]. The MAPK pathway is critical for oncogenic signalling and results from the activation of growth factor receptors, leading to cell proliferation and survival. The MAPK pathway is initiated with the activation of RAS, which then stimulates the downstream kinases RAF, the MAPK kinase (MEK1/2) and extracellular signal-regulated kinase (ERK1/2) [5] (online supplementary figure S1). Somatic activating BRAFV600E mutations are identified in LCH lesions in 30–60% of cases, including pulmonary LCH (PLCH) [6–12]. BRAF-activating mutations have generated interest in the use of BRAF inhibitor (BRAFi) treatment in progressive cases of LCH [5, 6, 13]. Importantly, the MEK/ERK pathway is activated independently of the lesion's BRAF status, indicating that other mechanisms could be involved in MAPK pathway activation in LCH [6]. More recently, mutations in MAP2K1 (a member of the MEK family) were identified in 15–50% of BRAF wild-type (WT) nonpulmonary LCH lesions, thereby explaining MAPK pathway activation in these cases [14–16]. Individual cases of other mutations in the MAPK pathway have also been reported [15, 17]. However, additional mechanisms remain to be identified to explain the MAPK pathway activation in the substantial subset of LCH lesions in which no mutation has been identified.

Mutations in the RAS family, small GTPases upstream of MEK/ERK, are able to activate the MAPK pathway [18]. We therefore screened for the presence of NRAS- and KRAS-activating mutations in a series of lung LCH biopsies, and compared the results with those obtained in nonpulmonary LCH lesions. Strikingly, whereas no NRAS mutations were present in the nonpulmonary LCH lesions, we identified NRASQ61K/R mutations in a significant subset of PLCH lesions. We also evaluated their functional consequences and sought an association with patient outcome.

Materials and methods

LCH biopsies

26 surgical lung biopsies from patients with PLCH were evaluated. The characteristics of these patients at diagnosis and during follow-up are presented in table 1. The outcomes of PLCH were assessed at the last available follow-up, and the patients were classified as improved or having persistent (stable) or progressive disease based on the variations of dyspnoea (assessed by New York Heart Association (NYHA) stage) and lung function as previously described [4]. No patient received any treatment for pulmonary LCH. 37 nonpulmonary LCH biopsies obtained from patients with isolated or multisystem disease were also studied. For five patients, two sites of biopsies were available (lung and another site n=4, bone and lymph node n=1). The characteristics of the entire study population of LCH patients are presented in online supplementary table S1.

View this table:
TABLE 1

Clinical characteristics of the 26 patients with pulmonary Langerhans cell histiocytosis whose surgical lung biopsies were analysed

The study was performed in accordance with the Helsinki Declaration and was approved by the INSERM Institutional Review Board and Ethics Committee in Paris (13-130), France. All patients provided informed consent.

Processing of LCH tissues

Formalin-fixed, paraffin-embedded (FFPE) biopsies were evaluated for the presence CD1a-positive nodules that were macrodissected for molecular biology analysis. To ensure that CD1a-positive cells were consistently present in the analysed specimens, CD1a immunostaining was performed every 10 consecutive tissue sections.

Lung tissue control

In cases in which the PLCH lesion was well circumscribed, the surrounding lung tissue containing <1% of CD1a-positive cells was evaluated as an internal control.

Grossly normal lung tissue from 10 patients who were smokers (six males, median (interquartile range (IQR)) age 64 (59–68) years) was obtained at the time of thoracic surgery for localised lung carcinoma (adenocarcinoma n=9, squamous cell carcinoma n=1) and was also analysed. Under light microscopy, these specimens exhibited mild fibrotic changes in the alveolar walls, accumulation of pigment-laden macrophages and alveolar epithelial hyperplasia.

Molecular analysis

DNA was extracted using the QIAamp DNA FFPE Tissue Kit for FFPE tissue from five 10-µm sections (Qiagen, Les Ulis, France) according to the manufacturer's protocol. DNA was qualified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and quantified using a Qubit® 2.0 fluorometer (Life Technologies, Saint-Aubin, France).

BRAF, NRAS and KRAS genotyping was performed using pyrosequencing, high-resolution melting (HRM) and enhanced ice-COLD-PCR (improved and complete enrichment coamplification at lower denaturation temperature PCR; E-ice-COLD-PCR) [19, 20]. MAP2K1 genotyping was performed using Sanger sequencing [14].

Pyrosequencing

Mutation detection, identification and quantification were performed by genotyping using a PyroMark-Q96 MD pyrosequencer (Qiagen, Hilden, Germany). PCR products were purified and rendered single-stranded using a PyroMark-Q96 Vacuum Workstation (Qiagen) [19].

High-resolution melting

BRAF and NRAS HRM analyses were conducted according to the manufacturer's protocol using an LC480 HRM Scanning Master on a LightCycler 480 (Roche, Boulogne-Billancourt, France) [19].

E-ice-COLD-PCR

The sensitive detection of mutations in NRAS codons 12, 13 and 61, BRAF codon 600, and KRAS codons 12 and 13 by E-ice-COLD-PCR was performed in a LightCycler 480 (Roche Applied Science, Penzberg, Germany). The reaction contained 20 nM BRAF V600, 50 nM NRAS Q61 or 40 nM KRAS G12 and G13 blocker probes [19–21]. For NRAS G12 and G13 assay, 200 nM forward (AATTAACCCTGATTACTG) and reverse (biotin-GGATCATATTCATCTACAA) primers, 2 µM SYTO9 and 20 nM blocker probe (GCGCTTTTCCCAAC+A+C+C+A+C+CTGCTCCAAC-phosphate) were used.

Mutation detection, identification and quantification were performed by pyrosequencing. The mutant allele frequency (i.e. relative frequency of an allele) was expressed as a percentage. The limit of detection was evaluated at 0.1% of the mutant allele. The percentage of mutation of each sample was estimated after mutation enrichment through E-ice-COLD-PCR using a standard curve including the following fractions of mutations: 5%, 1%, 0.5% and 0.1% (cell lines used: G-361 for BRAF, SW480 for KRAS, HL-60 for NRAS codon 61 and THP-1 for NRAS codon 12/13).

Sanger sequencing

MAP2K1 genotyping was performed using bidirectional Sanger sequencing of exons 2 and 3, as described by Brown et al. [14]. DNA was sequenced using a BigDye Terminator version 1.1 sequencing kit (Applied Biosystems, Foster City, CA, USA) and a 3130xl DNA analyser (Applied Biosystems).

Immunohistochemistry

The following monoclonal antibodies were used in this study: anti-CD1a (clone O10; Dako, Eching, Germany), anti-BRAFV600E (clone VE1; Spring Biosciences, Pleasanton, CA, USA), anti-phospho-ERK (pERK)-1/2 (clone MAPK-YT; Sigma, Lyon, France) and anti-phosphorylated protein kinase B (phospho-AKT) (Ser473) (clone D9E-XP; Cell Signaling, Saint Quentin Yvelines, France). The recently described NRASQ61R antibody (clone SP174; Spring Biosciences) was used to assess the expression of NRASQ61R protein [22]. BRAFV600E- and NRASQ61K/R-mutated melanomas were used as positive controls.

Immunoglobulin isotype controls were used to assess nonspecific binding. Immunohistochemistry (IHC) was performed on serial sections as previously described [23] using Vectastain ABC-alkaline phosphatase or peroxidase systems (Vector, Burlingame, CA, USA). For pERK expression, a semiquantitative staining score, graded from – (absent) to +++ (strongly positive), was derived for each sample by comparing the staining intensity of CD1a-positive cells to the most intensely stained melanoma used as a positive control. The VE1 antibody staining was scored positive when viable tumour cells showed a clear cytoplasmic staining. Isolated nuclear staining, weak staining of single interspersed cells, faint diffuse staining or staining of monocytes/macrophages was considered negative [24]. The immunostaining scoring was performed by an experienced pathologist (V.M.) who had no knowledge of the mutational status of the lesions.

Immunofluorescence

Anti-CD1a antibody was applied to FFPE PLCH tissue sections followed by Alexa Fluor 568-conjugated donkey anti-mouse antibody (Life Technologies). Subsequently, anti-NRASQ61R antibody was applied, followed by Alexa Fluor 488-conjugated goat anti-rabbit antibody (Life Technologies). Nuclei were counterstained using 4,6-diamidino-2-phenylindole (DAPI; Vector). Images were obtained using laser scanning confocal microscopy (Leica-Lasertechnik, Heidelberg, Germany).

In situ proximity ligation assay

Tissue sections were subjected to in situ proximity ligation assay (PLA) using a Duolink Detection kit (Olink Bioscience, Uppsala, Sweden) according to the manufacturer's instructions as previously described [25]. Briefly, slides were blocked, incubated with antibodies directed against CRAF (CliniSciences, Nanterre, France) and BRAF (Santa Cruz Biotechnology, Nanterre, France), and thereafter incubated with PLA probes, which are secondary antibodies (anti-mouse and anti-rabbit) conjugated to unique oligonucleotides. Circularisation and ligation of the oligonucleotides was followed by an amplification step. In this assay, a pair of oligonucleotide-labelled secondary antibodies (PLA probes) generates a signal only when bound in close proximity, thus allowing the detection of protein–protein interactions. Protein complexes were visualised as bright fluorescent signals (red dots) using a laser scanning confocal microscope (Leica-Lasertechnik). For quantification of PLA signals, the number of red dots per nuclei (DAPI signal) was counted (more than three fields) using ImageJ software (http://imagej.nih.gov/ij/) and a minimum number of three pictures per lesion were analysed [26].

Statistical analysis

Descriptive summary statistics, i.e. percentages for categorical variables, mean±sd or median (IQR) values for quantitative variables, were computed. A Kruskal–Wallis test was performed to compare the distribution of PLA dots according to mutation status. The comparison of patient outcome category (improved, stable or progressive disease) according to persistent or smoking cessation during follow-up was performed using the exact Fisher test.

The predictive value of patient characteristics at diagnosis, including BRAF and NRAS mutations, on patient outcomes (distinguishing improvement versus nonimprovement) was assessed by univariate and multivariable logistic regression models, where the strength of the association between each characteristic and the outcome was measured by the odds ratios (95% CI). Multivariable models included all variables associated with the outcome based on univariate analyses at the 10% level. Only those variables retained by a stepwise selection procedure were selected, at the 5% level.

All statistical analyses were performed using SAS version 9.3 (SAS, Cary, NC, USA) and R version 3.0.2 (www.R-project.org).

Results

BRAF and MAP2K1 status of PLCH lesions

The characteristics of the PLCH patients enrolled in the study are presented in table 1. BRAFV600E was detected by pyrosequencing and E-ice-COLD PCR in 13 (50%) out of 26 of the samples within CD1a-positive cell-enriched areas (table 2). BRAFV600E protein was also identified by IHC in 11 (85%) out of 13 BRAFV600E-mutated cases detected by genotyping (figure 1 and table 2).

FIGURE 1
FIGURE 1

Immunostaining on serial tissue sections of the same specimens. Expression of CD1a and VE1 in a BRAFV600E-mutated (patient 1) and in a BRAF wild-type (WT) (patient 17) pulmonary Langerhans cell histiocytosis (PLCH) specimen. Scale bar: 20 µm. 26 PLCH lesions were analysed.

View this table:
TABLE 2

Phospho-extracellular signal-regulated kinase (pERK) and BRAF/NRAS/MAP2K1 mutation status in the 26 analysed pulmonary Langerhans cell histiocytosis lesions

As MAP2K1 mutations that were mutually exclusive with BRAFV600E mutation were identified in nonpulmonary LCH lesions [14–16], we tested for the presence of these mutations in PLCH lesions. Among the 13 BRAF WT PLCH lesions, three (23%) specimens exhibited the MAP2K1C121S mutation (table 2).

The MAPK pathway was activated in all analysed cases, although at a variable intensity, as assessed by pERK IHC (table 2 and online supplementary figure S2).

NRAS mutations in PLCH lesions

Given that MAPK pathway activation was also observed in BRAF and MAP2K1 WT tissues, we evaluated whether PLCH lesions contain somatic mutations in the NRAS or KRAS oncogenes, potentially explaining MAPK activation. The 26 PLCH lesions were screened for NRAS G12/13 and Q61 and KRAS G12/13 mutations using pyrosequencing and HRM analysis. Among the 26 PLCH lesions, four cases exhibited a NRASQ61R/K mutation. Using E-ice-COLD-PCR, clonal NRAS Q61 mutations were identified in seven additional cases, which were NRAS WT using standard techniques. Thus, NRAS mutations were observed in 11 (42%) out of 26 PLCH lesions (table 2). In contrast, none of the PLCH lesions exhibited a NRAS G12 or G13 mutation, and only one lesion was positive for the KRAS G12S mutation (patient 15), although at the limit level of detection by E-ice-COLD-PCR.

For six PLCH specimens, the LCH lesion was well circumscribed and the surrounding lung tissue contained <1% CD1a-positive cells. Among these specimens, two PLCH lesions harboured a NRASQ61K and a NRASQ61R mutation, respectively. Interestingly, no NRAS mutation was detected in the surrounding lung tissue of these two specimens, even by E-ice-COLD-PCR.

To further demonstrate that the NRASQ61R mutation was specifically harboured by LCH CD1a-positive cells, we performed serial immunostaining on sections as well as double immunofluorescence and confocal microscopy with the recently described antibody against NRASQ61R protein [22]. As shown in figure 2, we could demonstrate specific NRASQ61R protein immunostaining by CD1a-positive cells in NRASQ61R-mutated PLCH lesions.

FIGURE 2
FIGURE 2

Expression of NRASQ61R protein in NRASQ61R-mutated pulmonary Langerhans cell histiocytosis (PLCH) lesions. a) Expression of CD1a and b) immunostaining with anti-NRASQ61R protein antibody performed on serial tissue sections. Scale bar: 30 µm. c) Confocal microscopy images of immunofluorescence staining of an NRASQ61R-mutated PLCH lesion stained with 4,6-diamidino-2-phenylindole (DAPI) (blue), anti-CD1a (red) and anti-NRASQ61R antibody (green), and a merged image of all three stains. Scale bar: 20 µm. All panels show representative images of the two NRASQ61R-mutated lesions (patients 7 and 10).

As NRAS mutations were not described in nonpulmonary LCH lesions, we tested for the presence of these mutations in 19 bone, 13 skin and five lymph node LCH biopsies. NRAS mutations were not detected in any case, not even by E-ice-COLD-PCR. In contrast, BRAFV600Emutations were identified in 38% of these specimens. Strikingly, among the four patients for whom both a lung biopsy and a nonpulmonary LCH tissue specimen were available, one patient (patient 25; table 2) harboured an NRASQ61K mutation in his PLCH lesion, whereas his cutaneous LCH lesion was NRAS WT.

Finally, because PLCH occurs almost exclusively in smokers, we also genotyped 10 control lung tissue specimens from patients who were smokers to evaluate the role of smoking per se on the eventual presence of NRAS mutations in the lung. No NRAS mutations were detected in any of these specimens.

Concurrent BRAFV600E and NRASQ61K/R mutations in pulmonary LCH lesions

Among the 11 NRAS-mutated PLCH lesions, seven harboured both BRAFV600E and NRASQ61K/R mutations with observed allele frequencies favouring the presence of clonal heterogeneity in these lesions (table 2). In contrast, no MAP2K1 concurrent mutation was observed in the NRAS-mutated PLCH lesions (table 2).

To further examine whether BRAFV600E and NRASQ61K/R concurrent mutations found in the same PLCH specimens were the result of different histiocyte clones, we analysed a second lung biopsy block in five of these cases by separately genotyping each CD1a-positive area. In these cases, each focal CD1a-positive area within the same specimen harboured either BRAFV600E or NRASQ61K/R alone, or both mutations (figure 3 and table 3). Interestingly, in three of the five cases, whereas NRASQ61K/R mutations were initially detected only by E-ice-COLD-PCR, these mutations were identified by pyrosequencing in separately genotyped CD1a-positive areas (tables 2 and 3). These findings strongly suggest that the concurrent BRAFV600E and NRASQ61K/R mutations identified in seven of 26 (27%) PLCH lesions are subclonal (i.e. not present in the entire population of tumour cells) and are carried by different CD1a-positive clonal populations.

FIGURE 3
FIGURE 3

BRAFV600/NRAS Q61 clonal heterogeneity in pulmonary Langerhans cell histiocytosis (PLCH) lesions. Different CD1a-positive areas were separately genotyped for BRAF and NRAS mutations (patient 10). Pyrograms of BRAFV600E and NRASQ61R mutations. Each sequence is derived from the corresponding lesion area. The different CD1a-positive areas from three PLCH lesions (patients 7, 10 and 12) were analysed. Scale bar: 1.5 mm.

View this table:
TABLE 3

Genotyping of separate CD1a-positive areas in pulmonary Langerhans cell histiocytosis specimens harbouring concurrent BRAFV600E and NRASQ61K/R mutations

Functional consequences of NRAS mutations in PLCH lesions

NRAS-activating mutations have been shown to induce BRAF and CRAF heterodimerisation, leading to MAPK pathway activation [27]. Using in situ PLA with a combination of CRAF and BRAF antibodies, we could detect a strong heterodimerisation (expressed as median (IQR) number of red dots per nuclei) in PLCH lesions harbouring NRASQ61K/R mutations: either alone (32 (21–41); n=3, patients 22, 23 and 25) or concurrent with BRAFV600E mutation (38 (30–47); n=3, patients 3, 4 and 7), as compared with BRAFV600E/NRAS WT (11 (3–14); n=3, patients 1, 5 and 6) or BRAF WT/NRAS WT (2 (2–3); n=3, patients 15, 17 and 20) lesions (p<0.0001; figure 4 and online supplementary figure S3).

FIGURE 4
FIGURE 4

In situ proximity ligation assay (PLA) demonstrating BRAF–CRAF dimerisation in an NRASQ61K-mutated lesion (patient 23). BRAF–CRAF heterodimerisation was visualised as red dots by in situ PLA and was detected with a fluorescent microscope; cell nuclei were stained with 4,6-diamidino-2-phenylindole (blue). Scale bar=10 µm.

As oncogenic RAS is a potential phosphatidylinositide 3-kinase/AKT signalling axis activator [28, 29], we assessed by IHC the AKT status in four PLCH lesions harbouring an NRAS Q61 mutation alone. AKT pathway activation was observed in all analysed cases, although with variable intensity (online supplementary figure S4).

Association of NRAS mutations with clinical outcomes

The amount of smoking (pack-years) did not correlate with the presence of NRAS mutations (p=0.67). 12 PLCH patients (46%) stopped smoking during follow-up (table 1). However, although a higher proportion of patients who stopped smoking improved, this difference in the distribution of the outcomes of PLCH according to smoking status during follow-up was not statistically significant (p=0.24).

In the univariate analyses, there was no association between the presence of BRAFV600E mutation alone and the patient outcome (p=0.24; table 4). Four variables were associated with the occurrence of spontaneous improvement of PLCH during follow-up at the 10% level, either positively for NYHA stage of dyspnoea (p=0.08) or negatively for forced expiratory volume in 1 s (FEV1) (p=0.10), the presence of an NRASQ61K/R mutation (p=0.05), and the presence of both NRASQ61K/R and BRAFV600E mutations (p=0.048). However, in the multivariate analysis, after incorporating the NYHA stage of dyspnoea and the FEV1 at baseline, the presence of both NRASQ61K/R and BRAFV600E mutations was no longer statistically significant (p=0.07).

View this table:
TABLE 4

Univariate analyses of the predictive factors at diagnosis of spontaneous improvement of pulmonary Langerhans cell histiocytosis during follow-up

Discussion

In the present study, we confirmed that the MAPK signalling pathway was constantly activated in PLCH lesions, as previously demonstrated in nonpulmonary LCH specimens [6]. This activation could be explained by the presence of the BRAFV600E somatic mutation in 50% of the lung LCH biopsies studied. This prevalence is in accordance with that observed in previous studies [6–12]. We also identified the presence of the MAP2K1C121S mutation, which may account for MAPK pathway activation in 20% of BRAF WT PLCH lesions.

The major finding of our study is the presence of recurrent NRASQ61K/R mutations (i.e. mutations occurring across multiple independent lesions) in 40% of PLCH specimens using both standard pyrosequencing and highly sensitive E-ice-COLD-PCR. It should be stressed that NRASQ61K/R mutations initially detected only by E-ice-COLD-PCR were easily identified by pyrosequencing when the genotyping was specifically focused on separate CD1a-positive areas, strongly suggesting that NRAS mutant alleles were harboured by PLCH cells. Consistently, using E-ice-COLD-PCR, we could not detect NRAS mutations in the surrounding lung tissue containing <1% CD1a-positive cells of NRASQ61K/R-mutated PLCH lesions. Finally, we clearly demonstrated that the NRASQ61R mutated protein was indeed located within CD1a-positive cells.

Although NRASQ61K/R somatic mutations were reported in Erdheim–Chester disease [30, 31], these mutations have not been previously identified in LCH lesions [6, 14, 15]. Consistently, we did not detect NRAS mutations in any of the nonpulmonary LCH lesions analysed, even by E-ice-COLD-PCR. Interestingly, in one patient, an NRASQ61K mutation was identified in the PLCH lesion, whereas no NRAS mutation was detected in his skin lesion using the same genotyping techniques. Taken together, the presence of NRASQ61K/R mutations appears to be a particular feature of LCH lung involvement.

The reason why the two previous studies that performed large targeted genotyping in lesions from patients with PLCH did not detect NRAS mutations remains unclear [6, 12]. In addition to the fact that a small number of cases were analysed, heterogeneity within PLCH lesions, as shown in our study, might explain these discrepancies.

Although PLCH occurs almost exclusively in smokers, smoking does not by itself explain the occurrence of NRAS mutations. Indeed, we did not detect these mutations in lung samples from smoking controls.

Another important finding of this study is the identification of concurrent NRASQ61K/R mutations in 50% of BRAFV600E-mutated PLCH lesions. Although BRAF and NRAS mutations are usually mutually exclusive, both mutations were recently identified in multiple myeloma and melanomas, supporting the clonal heterogeneity of these tumours [32–36]. When these two mutations were simultaneously detected, one was at a high frequency and the other at a low frequency [32], which was also the case in most analysed cases of our study. Here, we also demonstrated that BRAF and NRAS mutations could be present in different areas within the same lung biopsy, clearly demonstrating that different clones of cells harboured either NRAS and/or BRAF mutations. In this regard, whereas CD1a-positive cells infiltrating nonpulmonary LCH lesions were shown to be of clonal origin [37], Yousem et al. [38] demonstrated that these cells were polyclonal in most cases of PLCH. The results of the present study further support the polyclonal nature of LCH cells in PLCH.

In the univariate analyses, the patients whose lesions contained concurrent NRASQ61K/R and BRAFV600E mutations, but not an NRASQ61K/R or BRAFV600E mutation alone, exhibited poorer spontaneous outcomes. Additional studies on a larger series of PLCH patients are needed to confirm this finding, as this association was erased in the multivariate analysis.

The discovery of BRAFV600E in LCH lesions paved the way for the use of BRAFi (e.g. vemurafenib) treatment in progressive cases [13, 39, 40]. The fact that PLCH lesions with NRAS mutations exhibit activation of both MAPK and AKT pathways underlines the need for prudent use of BRAFi treatment in patients whose PLCH lesions are concomitantly mutated for both BRAF and NRAS, as resistance or paradoxical disease progression may occur [28, 29]. Resistance mechanisms acquired during BRAFi treatment have been shown to involve multiple signalling pathways, including NRAS mutations [28, 29]. In this regard, the co-occurrence of both BRAFV600E- and NRASQ61K-activating mutations was demonstrated in cells derived from patients with melanoma who experienced progressive disease under vemurafenib treatment [28]. In cells with sufficient levels of RAS activation, RAF forms activated dimers. BRAFi binding to one member of the RAF dimer results in the transactivation of the other and induces the activation of ERK signalling, thereby stimulating tumour proliferation [41].

In summary, this study provides further insights into MAPK pathway activation in LCH. Our findings also emphasise the importance of NRAS genotyping of PLCH lesions. Patients with a PLCH harbouring a NRAS mutation may benefit from MEK inhibitor (MEKi) treatment [42] or alternatively from a combined treatment with a BRAFi and MEKi in dual BRAF/NRAS-mutated cases, as recently suggested [43].

Acknowledgements

The authors thank Dr Giorgia Egidy-Maskos (INRA, Jouy en Josas, France) for pERK and pAKT IHC; Silvina Dos Reis Tavares, Aurélie Sadoux, Maeva Valluci and Farah Khayati (Hôpital Saint-Louis, Paris, France) for technical support; the Hôpital Saint-Louis Tumour Biobank for managing patient biological samples; and Elisabeth Savariau (Institut Universitaire d'Hématologie, Service d'Infographie, Hôpital Saint-Louis, Paris, France) for her assistance with the figures.

Footnotes

  • Editorial comment in Eur Respir J 2016; 47: 1629–1631.

  • This article has supplementary material available from erj.ersjournals.com

  • Support statement: The authors acknowledge support from Legs Poix (Chancellerie des Universités) and the Fonds de Dotation Recherche en Santé Respiratoire. Funding information for this article has been deposited with FundRef.

  • Conflict of interest: Disclosures can be found alongside the online version of this article at erj.ersjournals.com

  • Received October 9, 2015.
  • Accepted February 11, 2016.

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