Regular Article

MYELOID NEOPLASIA

Hematopoietic origin of Langerhans cell histiocytosis and

Erdheim-Chester disease in adults
Paul Milne,1 Venetia Bigley,1 Chris M. Bacon,2,3 Antoine Néel,4 Naomi McGovern,1 Simon Bomken,2 Muzlifah Haniffa,1
Eli L. Diamond,5 Benjamin H. Durham,5 Johannes Visser,6 David Hunt,7 Harsha Gunawardena,8 Mac Macheta,9
Kenneth L. McClain,10 Carl Allen,10 Omar Abdel-Wahab,5 and Matthew Collin1
1
Institute of Cellular Medicine and 2Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom; 3Department of
Cellular Pathology, Newcastle upon Tyne Hospitals National Health Service Foundation Trust, Newcastle upon Tyne, United Kingdom; 4Internal Medicine
Department, Hôtel-Dieu University Hopital, Nantes, France; 5Memorial Sloan Kettering Cancer Center, New York, NY; 6East Midlands Children’s and Young
Persons’ Integrated Cancer Service, Leicester Children’s Hospital, Leicester, United Kingdom; 7Medical Research Council Institute of Genetics and
Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom; 8Rheumatology Department, North Bristol National Health Service Trust, Bristol,

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United Kingdom; 9Blackpool Teaching Hospitals National Health Service Foundation Trust, Blackpool, United Kingdom; and 10Texas Children’s Cancer
Center, Baylor College of Medicine, Houston, TX

Langerhans cell histiocytosis (LCH) and Erdheim-Chester disease (ECD) are rare
Key Points
histiocytic disorders induced by somatic mutation of MAPK pathway genes. BRAFV600E
• Bone marrow progenitors, mutation is the most common mutation in both conditions and also occurs in the
monocytes, and myeloid DCs hematopoietic neoplasm hairy cell leukemia (HCL). It is not known if adult LCH or ECD
contain BRAFV600E alleles in arises from hematopoietic stem cells (HSCs), nor which potential blood borne precursors
adults with LCH and ECD. lead to the formation of histiocytic lesions. In this study, BRAFV600E allele–specific
polymerase chain reaction was used to map the neoplastic clone in 20 adults with LCH,
• Mutant allele distribution is
ECD, and HCL. BRAFV600E was tracked to classical monocytes, nonclassical monocytes,
not disease specific, but
and CD1c1 myeloid dendritic cells (DCs) in the blood, and mutations were observed
precursors have distinct in HSCs and myeloid progenitors in the bone marrow of 4 patients. The pattern of
LCH-like and macrophage involvement of peripheral blood myeloid cells was indistinguishable between LCH and
differentiation capacities. ECD, although the histiocytic disorders were distinct to HCL. As reported in children,
detection of BRAFV600E in peripheral blood of adults was a marker of active multisystem
LCH. The healthy counterparts of myeloid cells affected by BRAF mutation had a range of differentiation potentials depending on
exogenous signals. CD1c1 DCs acquired high langerin and CD1a with granulocyte-macrophage colony-stimulating factor and
transforming growth factor b alone, whereas CD141 classical monocytes required additional notch ligation. Both classical and
nonclassical monocytes, but not CD1c1 DCs, made foamy macrophages easily in vitro with macrophage colony-stimulating factor
and human serum. These studies are consistent with a hematopoietic origin and >1 immediate cellular precursor in both LCH and ECD.
(Blood. 2017;130(2):167-175)

Introduction
Langerhans cell histiocytosis (LCH) and Erdheim-Chester disease alleles in CD141 and CD11c1 peripheral blood populations.5 A
(ECD) are rare inflammatory myeloid neoplasms principally caused by hematopoietic origin was corroborated in 2 cases of MS-LCH through
mutations in the MEK–extracellular signal-regulated kinase (ERK) expansion of hematopoietic clones in vitro.5 These studies support a
signaling pathway, most commonly involving BRAF.1-5 LCH has a model in which MS-LCH arises in the hematopoietic stem cells (HSCs)
peak of incidence in childhood, but an increasing number of adults are and is disseminated to the periphery by myeloid cells carrying somatic
now also diagnosed, including some with a poor outcome because of mutation.13 However, the identity of blood-borne cells that give rise
multisystem LCH (MS-LCH).6-8 ECD, once considered a form of to peripheral tissue histiocytes remains open to further investigation,
“non-Langerhans” histiocytosis, appears to be a related condition by because .1 cell type carries BRAFV600E and myeloid lineages may
virtue of a common genetic landscape and the histological evidence have overlapping differentiation potentials.
of coexistent LCH and ECD in a proportion of adult patients.9,10 In Human peripheral blood contains several subsets of myeloid
contrast with LCH, ECD is rarely seen in children and predominantly mononuclear cells. Monocytes comprise ;10% of mononuclear cells
affects older males.11,12 and may be divided into classical, nonclassical, and intermediate on the
In children with MS-LCH caused by BRAFV600E allele–specific basis of CD14 and CD16 expression.14 All human monocytes express
polymerase chain reaction (PCR) has been used to demonstrate mutant CD11c. CD141 classical monocytes are the most numerous and can be

Submitted 16 December 2016; accepted 24 April 2017. Prepublished online as The publication costs of this article were defrayed in part by page charge
Blood First Edition paper, 16 May 2017; DOI 10.1182/blood-2016-12-757823. payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2017 by The American Society of Hematology

BLOOD, 13 JULY 2017 x VOLUME 130, NUMBER 2 167

168 MILNE et al BLOOD, 13 JULY 2017 x VOLUME 130, NUMBER 2

differentiated into macrophages and dendritic cells (DCs) in vitro. Flow cytometry
Blood DCs comprise 1% to 2% of mononuclear cells and are sub- Absolute quantitation was performed using TruCount tubes on whole blood with
divided into CD1231 plasmacytoid DCs and 2 subsets of CD11c1 a single-step lyse no-wash protocol. For more detailed analysis, whole blood was
myeloid DCs expressing CD1c1 and CD1411.15 CD1c1 DCs are separated by density centrifugation into mononuclear and red cell/neutrophil
the major population of myeloid DCs in blood and tissues. They may fractions. The red cell pellet was subsequently used for extraction of neutrophil
express langerin in vivo16 and readily acquire a Langerhans cell–like DNA. Flow cytometry was performed on an LSRII or LSR Fortessa X20
phenotype in vitro.17,18 cytometer from Becton Dickinson (BD; http://www.bd.com), and data were
The differentiation potentials of monocytes and blood DCs overlap, analyzed with FlowJo (Treestar). Fluorescence-activated cell sorting was
and monocyte-derived DCs bear many markers in common with performed using a BD FACS Aria. Sorted cells were collected into microfuge
CD1c1 myeloid DCs.19 Monocytes have plasticity in many directions tubes containing RPMI with 10% fetal calf serum.
including DC and macrophage axes20,21 and may also express langerin Antibodies were from BD unless stated otherwise and are denoted as
follows: antigen fluorochrome (clone) CD1a BV421 (HI149; Biolegend; http://
in vitro under certain conditions.22 Thus multiple blood-derived
www.biolegend.com); CD1c APC (AD5-8E7; Miltenyi Biotec; http://www.
populations have the potential to contribute to histiocytic lesions. miltenyibiotec.com); CD3 FITC (SK7); CD3 V500 (UCHT1); CD7 FITC
The precursor populations of adults with LCH, ECD, or LCH/ECD (4H9); CD10 PE-TR (B-Ly6; Beckman Coulter; http://www.beckmancoulter.
crossover disease remain undefined. It is not known whether MS-LCH

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com); CD11c A700 (B-ly6); CD14 APCCy7 (MFP9); CD14 FITC
in adults correlates with the presence of mutant alleles in peripheral (M5E2); CD16 FITC (NKP15/Leu-11a); CD16 PeCy7 (B73.1); CD19
blood or if it is possible to discern a similar risk stratification in patients FITC (4G7); CD19 PECy7 (SJ25C1); CD20 FITC (L27); CD34 PE (8G12);
with ECD. NRAS mutation was detected in the CD141 fraction of CD34 FITC (8G12); CD34 APC (581); CD38 PECy7 (HB7); CD45 V450
peripheral blood mononuclear cells (PBMCs) in 1 patient,23 and ERK (2D1); CD45 APCCy7 (2D1); CD45RA BV510 (HI100; Biolegend); CD56
activation was reported in 1% to 3% of monocytes of an ECD cohort.24 FITC (NCAM16.2); CD90 PerCPCY5.5 (5E10; Biolegend); CD123
However, the involvement of other myeloid lineages has not been PerCPCY5.5 (7G3); CD123 PE (9F5); CD207 PE (DCGM4; Beckman
Coulter); HLA-DR V500 (L243/G46-6); and HLA-DR A700 (L243/G46-6).
evaluated in ECD.
Recent RNA sequencing suggests that LCH lesions are enriched for BRAFV600E allele–specific PCR
DC gene transcripts, whereas biopsies of ECD are enriched for myeloid
precursor and macrophage gene expression.25 It has been suggested Genomic DNA from sorted blood cells, BM cells, control cell lines, or cell-free
that DC pathways of development may be involved in the generation sources was extracted using Qiagen micro-kits (Qiagen; http://www.qiagen.
of LCH, whereas monocyte-macrophage differentiation could give rise com). BRAF mutation and reference quantitative PCR was performed with
competitive allele-specific Taqman mutation detection assays: Mutation Allele
to ECD. Finally, BRAFV600E has been detected in HSCs of patients
Assay, BRAF_476_mu 4465804 Hs00000111_mu; Gene Reference As-
with hairy cell leukemia (HCL) and confirmed by xenografting.26 There
say, BRAF_rf - 4465807 Hs00000172_rf, according to the manufacturer’s
does not appear to be a clinical overlap between HCL and histiocytosis, instructions (Life Technologies; http://www.lifetechnologies.com). A standard
and the pathogenesis of these 2 different conditions from a common curve was derived from 6 serial dilutions of genomic DNA from BRAFV600E
somatic mutation is poorly understood. HCL is therefore an interesting melanoma cell line A375 and Epstein-Barr virus–transformed lymphoblastoid
disease to evaluate in parallel and provides a useful control for the cell lines; 0%, 0.1%, and 10% V600E mutation controls were performed with
specificity of allele mapping in histiocytosis. every run. The percentage of BRAFV600E mutation was determined from the dCT
In this report, we have analyzed the distribution of BRAFV600E in (CT reference 2 CT mutant) using the standard curve. Graphs were plotted with
the peripheral blood and bone marrow (BM) progenitor compartments Prism Version 5.0 (GraphPad Software Inc.).
of adults with MS-LCH, LCH/ECD crossover, and ECD. We sought
to determine whether mutant alleles could be tracked to the HSCs In vitro differentiation
and whether there was a correlation between the architecture of the Aliquots of 10 000 sorted cells from healthy controls were cultured in RPMI
neoplastic clone and the clinical phenotype of histiocytosis. We also 1640 with 10% fetal bovine serum or 5% human serum in 100 mL in 96-well
aimed to intersect these observations with studies to determine the plates. Supplements were added: granulocyte-macrophage colony-stimulating
potential of DC and monocyte lineages to generate LCH-like cells factor (GM-CSF), 50 ng/mL; transforming growth factor b (TGF-b), 10 ng/mL;
and foamy macrophages as in vitro surrogates of pathological macrophage colony-stimulating factor (M-CSF), 50 ng/mL. Cultures were
histiocytes. maintained for 3 days before analysis by flow cytometry. For phase-contrast
microscopy, cells were cultured in Thermo Scientific Nunc chamber slides
system for 7 days, washed gently, and fixed with 1% paraformaldehyde in
phosphate-buffered saline, and images were acquired with Carl Zeiss Axioplan
2 340 objective and Carl Zeiss Axiovision software release 4.8.
Methods
Statistical methods
Patient samples, lesional BRAFV600E testing, and clinical data
Mann-Whitney U tests, Student t tests, and Fisher’s exact test were performed
Blood, skin, BM, and surplus biopsy material were obtained from LCH and ECD using GraphPad Prism 5.0 software.
patients referred to a local clinic with ethical approval from the Newcastle and
North Tyneside Research Ethics Committee. Genomic DNA from formalin-
fixed paraffin-embedded LCH lesions was extracted using Promega Maxwell
Tissue DNA Purification Kits (Promega; http://www.promega.co.uk), and the
region flanking codon 600 amplified by PCR. Amplicons were purified and Results
genotyped by primer extension for the c.1799T.A, p.Val600Glu (V600E) using
the Sequenom iPlex protocol (Sequenom; http://www.sequenom.com). The
Fifty-two adults with histiocytosis and 4 with HCL were recruited
extension products were detected by matrix-assisted laser desorption/ionization from a local clinic and collaborating centers (Figure 1). Twenty with
time of flight (MALDI-TOF) mass spectrometry using a Sequenom Typer 4.0. histiocytosis and all with HCL had confirmed BRAFV600E lesions.
Disease activity was assessed by radiological examination including magnetic Three additional children with BRAFV600E-mutated LCH were
resonance imaging and positron emission tomography. included in some analyses for comparison. Further clinical details
BLOOD, 13 JULY 2017 x VOLUME 130, NUMBER 2 HEMATOPOIETIC ORIGIN OF ADULT HISTIOCYTOSIS 169

Figure 1. Patients. Summary of adult patients included in the study.

Asterisks indicate the number of patients with BRAFV600E detectable
in PBMCs. The BRAF wild-type (WT) ECD group includes 1 patient
Adult Histiocytosis HCL
with RAS mutation. NT, not tested. 52 4

Lesion Lesion
BRAFWT /NT BRAFV600E
32 20

LCH ECD LCH LCH/ECD ECD

27 5 8 (4*) 4 (3*) 8 (4*)

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are given in supplemental Table 1 (available on the Blood Web In contrast to patients with HCL, in which BRAFV600E was largely
site). confined to the HLA-DR1lineage1 quadrant containing hairy cells,
Detection of BRAFV600E was performed using an allele-specific mutation in patients with histiocytosis was highly enriched in the
PCR and was sensitive to 0.01% with a quantitative range to 0.1% HLA-DR 1 lineage 2 quadrant containing monocytes and DCs
(Figure 2A). BRAFV600E alleles were detectable in DNA isolated from (Figure 3A). Some overspill into adjacent quadrants was observed,
PBMCs of patients with active MS-LCH with or without coexistent owing to the close gating. For example, in histiocytosis patients,
ECD (LCH/ECD crossover) but were only present in 1 patient with positivity appeared in the HLA-DR2 lineage2 quadrant. This was
single-system (SS) LCH (Figure 2B). ECD is not conventionally classified further studied in case A2951 and found to be associated with
into SS or MS. Nearly all cases involved multiple systems. There was no CD141 monocytes and CD11c1 myeloid precursors expressing low
clear correlation between clinical features and BRAFV600E detection HLA-DR at the top of the double-negative gate (data not shown).
in the PBMCs (see supplemental Table 1 for details). The mononuclear compartment was further fractionated to explore
A simple linear correlation was observed between the frequency the distribution of mutant alleles at higher resolution (supplemental
of mutant allele detection in PBMCs vs cell-free plasma DNA, Figures 1 and 2; Figure 3B). As expected, BRAFV600E was localized to
with approximately half-log greater sensitivity for detection in cellular hairy cells with a low level of alleles detectable in phenotypically
DNA (Figure 2C). In order to exclude the uptake of free DNA into the normal B and NK cells but none in the remaining monocytes and
cell-associated pool by phagocytosis or macropinocytosis, purified myeloid cells. In contrast, the majority of alleles in patients with
BRAFV600E-mutated DNA from A375 cells was incubated with whole histiocytosis were found in CD141 classical monocytes, CD161
blood of healthy controls (Figure 2Di), and sorted cell fractions were nonclassical monocytes, and CD11c1CD1c1 myeloid DCs. Plasma-
cultured with medium containing 20% serum from a patient with HCL cytoid DCs harbored some alleles in LCH patients, and in 1 case,
(Figure 2Dii). Cellular and cell-free DNA was then recovered and BRAFV600E was also detectable at a low level in B and T cells. The
subjected to PCR. Mutated BRAF alleles were only recovered from the distribution of alleles did not vary significantly between LCH, ECD,
cell-free fraction, but not the cellular DNA, indicating that uptake of and patients with LCH/ECD. In addition to these compartments, we
exogenous DNA did not account for cell-associated BRAFV600E. With studied CD141CD161 intermediate monocytes and CD11c1CD1c2
patient serum, cell cultures not only remained negative but diluted or myeloid cells in a subset of patients (see supplemental Figures 1, 2,
degraded the cell-free signal. and 4 for details). These did not differ significantly from the allele level
The pattern of mononuclear cells observed by flow was analyzable in CD141 classical monocytes and CD1c1 myeloid DCs, respectively.
by conventional gating, and no atypical populations were detected BRAFV600E mutation was previously reported in children with
(supplemental Figure 1). Neither langerin1CD1a1 LCH-like cells nor MS-LCH, using a more simple gating strategy to obtain CD141 and
CD1031CD11c1 hairy cells were detectable in the peripheral blood CD11c1 myeloid cells.5 CD11c1 myeloid cells comprise mul-
of any of the histiocytosis patients (supplemental Figure 1). Absolute tiple fractions including CD1c1 myeloid DCs, CD161 nonclassical
counting of PBMCs from patients with histiocytosis revealed mono- monocytes, and CD11c1CD1c2 cells, which were analyzed separately
cytosis and increased numbers of total CD11c1 and CD11c1CD1c2 here. The relationship between these populations is summarized in
myeloid cells (supplemental Figures 1 and 2). There were no supplemental Figure 4.
detectable differences between BRAF-mutated and WT disease, In order to probe the hematopoietic origin of LCH and ECD,
although power to detect subgroup differences was limited. No BM was analyzed from 2 patients with MS-LCH and 2 with ECD. One
cytological atypia was observed among monocytes and DC subsets ECD patient (A7344) had mutated NRAS rather than BRAF and
(supplemental Figure 3). was previously reported to have a high level of NRAS mutation in
In order to map the distribution of mutant alleles, PBMCs from 9/11 CD141 peripheral blood monocytes (A7344).23 A commonly used
patients with circulating BRAFV600E and 2 children with MS-LCH were schema for immunophenotyping (supplemental Figure 5) revealed
fractionated into 4 quadrants according to the expression of HLA-DR that the common myeloid progenitor (CMP) and granulocyte-
and lineage (CD3, CD19, CD20, and CD56). The gates were completely macrophage progenitor (GMP) compartments were relatively expanded
apposed in order to account for all mutant alleles present in PBMCs and (Figure 4A). BRAFV600E was detectable in CD901CD45RA2CD38low
to exclude the inadvertent loss of signal to an uncharacterized disease- HSCs, CD38highCD45RA2 CMP, and CD45RA1 GMP (Figure 4B).
related population. The mutant allele was present at ,1% in HSCs and was most enriched
170 MILNE et al BLOOD, 13 JULY 2017 x VOLUME 130, NUMBER 2

A 30
B
100
p = 0.027
25
10

% BRAF V600E
dCT (CTmut - CTref)

20
1
POQR
15
0.1

10
Positive 0.01

5 0
SS MS MS LCH ECD HCL

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0 LCH LCH + ECD

% 0 0.01 0.1 1 10 100

BRAFV600E

C HCL LCH LCH/ECD

D
Cell-Free DNA
100 i. ii. Cellular DNA
r2 0.41 100
p 0.03
Free DNA % BRAF V600E

10
10
% BRAF V600E

1
1

0.1
0.1
0.01

0.01 0.001
0.01 0.1 1 10 100 Plasma PBMC No CD14+ CD16+ CD1c+
Cells Mono Mono mDC
Cell DNA % BRAF V600E

Figure 2. Detection of BRAFV600E alleles. (A) A standard curve constructed using DNA purified from a dilution series of BRAFV600E-positive melanoma cell line A375 into WT
Epstein-Barr virus–transformed lymphoblastoid cell line. The quantitative limit of detection was 0.1%; the absolute limit of detection was 0.01% (positive outside of quantitative
range [POQR]). (B) BRAFV600E allele frequency in bulk PBMCs in cases of lesion BRAFV600E 1 LCH, LCH/ECD, and ECD. Contingency of positive PBMCs upon MS-LCH
tested by Fisher’s exact test. (C) Correlation between mutated allele burden in cell-free plasma and PBMC DNA in LCH, ECD, and HCL. (Di) Test of exogenous free DNA
uptake by PBMCs. BRAF-mutated DNA (derived from melanoma cell line A375) was spiked into whole blood for 24 hours at room temperature. Plasma and PBMCs were
isolated by density centrifugation. DNA was extracted from both fractions and subjected to allele-specific PCR. (Dii) Test of exogenous free DNA uptake by sorted cells
incubated at 37°C for 24 hours in medium supplemented with 20% human serum from a patient with HCL. DNA from supernatants and cell pellets, as indicated, was subjected
to allele-specific PCR. mDC, myeloid dendritic cell.

in the CMP fraction. Mature histiocytes arising from the mutated clone cells with high langerin and CD1a contain Birbeck granules.17,18,22,28,29
were rigorously excluded in this analysis (supplemental Figure 5). A Previous reports are discordant and have not examined monocytes and
pediatric patient included for comparison showed a similar distribution DCs from peripheral blood and compared their differentiation capacity
of alleles. The NRAS-mutated patient was quantified by peak height in the same platform. We therefore tested the development of a
on Sanger sequencing at 21% to 63% in myeloid precursor fractions. Langerhans cell (LC)–like phenotype by blood monocytes and CD1c1
Progenitor cells from this patient were demonstrated to engraft in DCs using conditions that had been previously described in either
immunodeficient mice, in the accompanying paper.27 monocytes or DCs17,18,22,28,29: soluble factors GM-CSF and TGF-b
These results suggest that LCH and ECD arise in the HSCs and and the notch ligands d-like protein 1 (DLL1) and DLL4, expressed
are transmitted to peripheral tissues by blood-borne myeloid cells. on mouse OP9 cells. In order to study the potential to develop foamy
However, there was no obvious disease-specific bias in the distribution macrophage morphology, we used M-CSF with 5% human serum.
of mutant alleles among myeloid cells that might correlate phenotypic As shown previously, CD1c1 DCs attained high langerin
differences between LCH and ECD. We therefore evaluated the expression with GM-CSF and TGF-b alone. Soluble factors GM-
potential of each myeloid fraction to give rise to the characteristic cells CSF and TGF-b were not sufficient to induce high langerin in
of LCH and ECD lesions through in vitro differentiation of sorted CD141 monocytes, but this potential was revealed by coculture with
fractions from healthy controls. notch ligands DLL1 and DLL4. CD161 monocytes did not express
It has previously been demonstrated that monocytes and CD1c1 high langerin under any conditions, but notch ligation also further
DCs can express langerin in vitro under different conditions, but only enhanced the expression of langerin by CD1c1 DCs in response to
BLOOD, 13 JULY 2017 x VOLUME 130, NUMBER 2 HEMATOPOIETIC ORIGIN OF ADULT HISTIOCYTOSIS 171

A
A2123 A7044 A7073 A7264
1% 23% 11% 32%

HCL
1%
99% 77% 88% 68%

A7646 A7908
A2951 A7495 (child) (child)
LCH 22% 3% 6% 29%
2% 1% 2%
Live Cells
10% 6%
78% 85% 93% 93%
HLADR

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A2712 A7502 A7791
LCH & ECD

6% 12% 14%
Lineage
CD3,19,20,56 3%
20%
94% 88% 63%

A7343 A7348 A7349 A7640

2%* 1%*
ECD

4% 3%
0.5%* 14%
100% 82% 97% 100%

B
CD14+ CD16+ CD1c+ CD123+
Neuts T B NK HCL
Mono Mono mDC pDC

A2123 0.00 0.00 0.00 0.00 0.00 0.00 0.05 0.40 22.85
A7044 0.00 0.00 0.00 0.00 0.00 0.00 0.35 1.06 73.65
HCL
A7073 0.00 NT NT NT NT 0.01 26.87 0.10 38.21
A7264 0.00 0.00 0.00 0.00 0.00 0.03 0.29 0.01 29.34

A2951 0.00 4.82 45.61 4.96 0.90 0.03 0.16 0.00 N/A
A7495 0.00 17.74 39.48 7.94 1.04 0.00 0.00 0.00 N/A
LCH
A7646* 0.00 1.70 2.66 1.15 0.22 0.00 0.00 0.00 N/A
A7908* 0.00 20.07 41.75 13.75 1.88 0.00 0.00 NT N/A

A2712 0.00 0.14 4.07 0.11 0.06 <0.01 0.00 0.00 N/A
LCH/ECD A7502 0.00 0.17 0.20 0.08 0.00 0.00 0.00 0.00 N/A
A7791 0.00 0.20 0.71 0.23 0.00 0.00 0.00 0.00 N/A

A7348 NT 0.38 1.36 1.63 0.00 0.00 0.00 0.00 N/A

A7349 NT 1.42 0.47 1.06 0.00 0.00 0.00 0.00 N/A
ECD
A7640 0.00 0.08 0.16 0.00 0.00 0.00 0.00 0.00 N/A
A7343 NT 0.00 0.07 0.00 0.00 NT 0.00 0.00 N/A

Figure 3. Allele-specific PCR of BRAFV600E in PBMCs. (A) The distribution of BRAFV600E in PBMCs sorted into quadrants as shown according to expression of HLA-DR
and lineage. Pie charts summarize the distribution of mutant alleles in HCL, LCH, and ECD. The area of each quadrant in the pie is proportional to the total number of mutant
alleles in that quadrant (ie, the percentage positivity of the quadrant multiplied by the number of cells in the quadrant). All patients had active disease at the time of sampling.
Two children with LCH included for comparison are indicated. Asterisk indicates the threshold of detection for a negative result where cell numbers were limiting. (B) The
distribution BRAFV600E among peripheral blood cells showing the percentage of mutated alleles detected. Gray shading indicates positive fractions. Asterisk indicates child
with MS-LCH shown for comparison. All patients had active disease at the time of sampling. Italics indicate those who had received prior treatment. B, B cell; Mono, monocyte;
Neuts, neutrophil; NK, natural killer; NT, not tested owing to lack of or insufficient material; pDC, plasmacytoid dendritic cell; T, T cell.
172 MILNE et al BLOOD, 13 JULY 2017 x VOLUME 130, NUMBER 2

Figure 4. Mononuclear profile and BRAF allele

A LCH ECD frequency in CD341 BM progenitors. (A) Relative
proportions of progenitor fractions among CD341
A2712 A2951 A8086 A7791 A7344 BM mononuclear cells compared with healthy controls
(n 5 21), expressed as percentage of total live cells.
100 100 Error bars depict 95% confidence intervals of healthy
controls. Populations: B/NK, B/NK cell progenitors;
LMPP, lymphoid-primed multipotent progenitors; MEP,
% of live CD45+ cells

megakaryocyte–erythroid progenitor; MPP, multipotent

% of CD34+ cells

10 10 progenitor. (B) The distribution of BRAFV600E among

CD341 BM progenitors. Gray shading indicates posi-
tive fractions. BM aspirate was obtained in parallel
with the peripheral blood shown previously. *Child;
†
NRASQ61R Sanger sequencing. NT, not tested owing to
1 1 insufficient number of cells.

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0.1 0.1
CD34+ HSC CMP GMP LMPP BNK MEP MPP

B
HSC CMP GMP LMPP B/NK HCL
HCL A7264 0.57 0.07 0.05 33.37
A2712 0.47 4.56 0.61 0.00 0.00 N/A
LCH A2951 0.61 12.67 1.33 0.20 0.00 N/A
A8086* 0.15 0.29 0.85 0.05 0.03 N/A
A7791 0.10 0.35 0.48 0.01 NT N/A
ECD
A7344† 29.00 21.00 63.00 0.00 0.00 N/A

soluble factors (Figure 5; supplemental Figure 6). In macrophage negative). Although we localized most of the BRAFV600E alleles in
differentiation conditions, CD1c1 DCs were unable to form foam peripheral blood to the HLA-DR1lineage2 compartment of both LCH
cells, whereas both monocyte subsets developed this phenotype. A and ECD, some overspill was observed into the HLA-DR2lineage2
summary of the distribution of BRAF alleles and differentiation cells, owing to this strategy. Although a proportion of this signal was
potential of myeloid lineage affected is shown in Table 1. contained in HLA-DRloCD141 monocytes, CD11c1CD1c2 myeloid
progenitors that may generate macrophages are also found in this
region. In particular, ECD patients frequently have expanded
myelopoiesis with an element of left shift that mobilizes these cells into
Discussion the peripheral compartment; thus, we cannot exclude a contribution of
more primitive myeloid precursors to the lesions of ECD. Notably,
Prior to the discovery of BRAFV600E, there was accumulating evidence recent gene expression studies described enrichment for myeloid
that both LCH and ECD were clonal proliferations of histiocytes, progenitor signatures in ECD lesions.25
including a remarkable insight by Chetritt et al that ECD was the There is significant interest in cell-free DNA as a marker of
“macrophage counterpart to LCH.”30,31 Here we have exploited the neoplasia, and this is also detectable in histiocytosis.33 In our hands,
recurrent BRAFV600E mutation to map the neoplastic clones of LCH and plasma cell-free BRAFV600E was approximately half-log lower in
ECD. The results further reinforce the concept that LCH and ECD are abundance than cell-associated alleles. We considered the possibility
closely related hematopoietic neoplasms, regardless of their distinctive that exogenous DNA might account for the mutant alleles found in
pathological features.32 PBMCs, especially as the signal was localized to monocytes with phago-
A high-resolution analysis of myeloid cells demonstrated the cytic activity. However, exposure of healthy control blood to BRAF-
highest frequency of mutant alleles in CD141 classical monocytes, mutated genomic DNA did not result in a cell-associated signal.
CD161 nonclassical monocytes, and CD1c1 myeloid DCs. Un- Furthermore, the lack of mutated alleles in neutrophils, but the localization
expectedly, the nonclassical monocyte is a reservoir of BRAF mutation of mutation to nonphagocytic CD341 progenitor cells, argues against
and accounts for a proportion of the signal previously tracked to phagocytic uptake of exogenous DNA as a significant source of artifact.
CD11c1 myeloid cells.5 We also mapped BRAF mutation to cells Total RNA profiling reveals a strong DC-related and macrophage-
defined by phenotype as HSCs in LCH, ECD, and HCL. BRAFV600E related signature in LCH and ECD, respectively.25 However, there
has previously been reported in HSCs of children with MS-LCH5 and was no bias in the distribution of mutated alleles to suggest that
patients with HCL,26 and it remains unclear how the same mutation the phenotypic differences of LCH and ECD lesions are determined
present in HSCs generates such unrelated pathologies. Although there by the myeloid lineages harboring BRAF mutation. This may not be
is a documented overlap between LCH and ECD,9,10 HCL is unrelated surprising given the relatively high incidence of overlap cases now
in incidence, and we did not observe HCL leukocytes in any patient documented.9,10 Many alternatives may explain the variation between
with histiocytosis. LCH and ECD, such as genetic background, age-related myeloid
The initial cell-sorting approach adopted apposed gating of the hematopoietic bias,34 additional somatic mutation,35 or extrinsic inflam-
HLA-DR vs lineage plot of total PBMCs in order to account for all the matory signals arising independently in the local tissue environment.
mutated alleles present in PBMC DNA (neutrophils were consistently The difference in median age of onset between LCH and ECD is
BLOOD, 13 JULY 2017 x VOLUME 130, NUMBER 2 HEMATOPOIETIC ORIGIN OF ADULT HISTIOCYTOSIS 173

Figure 5. In vitro differentiation potential of mono-

cytes and myeloid DCs. (A) Sorted CD141 monocytes, A
CD161 monocytes, and CD1c1 myeloid DCs from healthy

Langerin
controls cultured for 3 days in conditions as shown.
LangerinhighCD1a1 gates depicted contain LCH-like cells
with Birbeck granules. Notch ligands DLL1 And DLL4 CD1a CD14+ CD16+ CD1c+
were presented on transfected mouse OP9 cells. Repre- Monocyte Monocyte DC
sentative data of 5 independent experiments. (B) Sorted 0.36% 0.25% 29.0%
CD141 monocytes, CD161 monocytes, and CD1c1 myeloid GM-CSF
DCs from healthy controls cultured for 7 days with M-CSF TGFβ
and 5% human serum (HS) to reveal potential to differen- (cell-free)
tiate into foamy macrophages. Representative phase
contrast images of 5 independent experiments.
2.10% 0.22% 36.6%
GM-CSF
TGFβ
OP9

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22.2% 0.32% 41.9%
GM-CSF
TGFβ
OP9-DLL1

65.9% 5.81% 49.3%

GM-CSF
TGFβ
OP9-DLL4

B CD14+ Monocyte CD16+ Monocyte CD1c+ DC

30μm

consistent with an influence of age-related myeloid bias or clonal The differentiation of myeloid cells in response to cytokine and
hematopoiesis. From a pathological perspective, the occurrence of LCH notch-mediated signaling showed that .1 precursor has the potential to
and ECD together in a single lesion suggests a cell-intrinsic form cells with characteristic properties of LCH and ECD histiocytes
mechanism.10 However, in cases where an overlap syndrome occurs (Figure 6). The critical role of notch signaling in permitting LC
with distinct and characteristic anatomical distributions of each disease, development from monocytes was first reported in response to
the tissue environment may direct either LCH- or ECD-like pathology, DLL1.22 Without this signal, CD141 classical monocytes only express
through extrinsic signals. In keeping with previous reports of expanded low langerin and are unable to form langerinhighCD1a1 cells containing
myelopoiesis in MS-LCH,36 we observed an increase in blood myeloid Birbeck granules in response to GM-CSF and TGF-b. In contrast,
cells and BM myeloid progenitors in adult LCH and ECD. CD1c1 DCs spontaneously express langerin in vivo16 and rapidly
acquire LC-like properties when exposed to GM-CSF and TGF-b.17,18
In vitro differentiation experiments presented here clearly show that 2
Table 1. Table summarizing BRAFV600E mutant allele frequency
pathways may generate langerinhigh cells and that DLL4 provides a
among blood monocytes and myeloid DCs (data from Figure 3) and
the potential of WT cells to form langerinhigh LCH-like cells or foamy more potent signal for monocytes than DLL1. Recent molecular studies
macrophages in vitro have shown that DLL1 induces LC development in monocytes by
CD14 CD16 CD1c downregulating KLF and depressing RUNX3.29 Elevated GM-CSF,
% BRAFV600E
M-CSF, and FLT3 ligand have been reported in the serum of patients
Median 0.38 1.36 0.2
with LCH, consistent with a pathological role of these cytokines.36,37
Range 0-18 0-45 0-8 Intrinsic notch signaling was previously suggested as a key mechanism
Langerin1 potential promoting LCH pathogenesis, through expression of Jagged 2
GM-CSF 1 TGF-b 2 2 11 by lesional histiocytes. 28 Taken together, these observations do
1DLL1 1 2 111 not support a simple dichotomous model that LCH arises from
1DLL4 111 2 111 the DC pathway and ECD from the monocyte-macrophage
Foamy mac potential 111 111 2 pathway of myeloid cell development. Classical monocytes
Frequency of langerin high
indicated as follows: 2, ,10%; 1, 10%-25%; 11, appear able to contribute to histiocytes of both disorders, depending
26%-35%; 111, .35%. on the signals they receive. CD1c1 DCs, on the other hand, were
174 MILNE et al BLOOD, 13 JULY 2017 x VOLUME 130, NUMBER 2

The finding that BRAF-mutated PBMCs are detected at higher

CD34+ levels in MS-LCH has potential utility as an indicator of clinical risk and
BM progenitors response to therapy, as suggested in children.5 Our conclusion in adults
(<1%) is based on only 8 patients and should be expanded to a larger cohort
ideally. The sensitivity of detection using allele-specific PCR is critical
HCL as less sensitive techniques failed to find BRAF mutation in periph-
eral blood or BM fractions previously.2,4 BRAFV600E was detected in
CD1c+ CD14+ CD16+ PBMCs of a subset of ECD patients, and it remains to be determined,
DC monocyte monocyte
using a large group, whether this segregates with disease risk in ECD.
Blood
(<3%)

GM-CSF + Notch M-CSF

TGFβ Acknowledgments

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The authors are grateful to Andrew Filby and the Newcastle
Lesion University Flow Cytometry Core Facility for assistance with the
(50-100%) generation of flow cytometry data, and Alex Laude and the Bioimaging
Selective advantage core for assistance with microscopy.
for BRAFV600E
LCH ECD This work was supported by a grant from Cancer Research UK
CD1a+ Langerin+ CD68+ CD163+
(C30484/A21025) and by the Histiocytosis Association and Bright
Red. V.B. is supported by a Wellcome Trust Intermediate Clinical
Figure 6. Potential precursor pathways in MS-LCH and ECD. Schema summarizing
potential precursor pathways in MS-LCH and ECD based on the distribution of
Fellowship (101155/Z/13/Z); M.H. is supported by a Wellcome
BRAFV600E alleles in peripheral blood and the differentiation potentials of healthy control Trust Senior Clinical Fellowship (WT088555MA); S.B. was
cells in vitro. A minority of cells (filled) in the BM and blood contain BRAF mutation. The supported by a National Institute for Health Research Clini-
principal observation is that ,1% clonal hematopoiesis gives rise to ,3% mutated blood
precursors that appear to be strongly selected for in peripheral tissue environments,
cal Lectureship; B.H.D. is supported by the American Society of
resulting in lesional histiocyte mutation levels of up to 100%. Lesions potentially consist Hematology Senior Research Training Award for Fellows and the
of .1 precursor as indicated by blue for CD1c1 DCs, red for classical monocytes, and New York State Council on Graduate Medical Education Empire
brown for nonclassical monocytes. The depiction of lesion composition is conjectural Clinical Research Investigator Program Fellowship; and E.L.D. and
and not based on experimental observation. Furthermore, additional contributions from
other, more primitive myeloid progenitors cannot be excluded at present. O.A.-W. are supported by grants from the Erdheim-Chester Disease
Global Alliance and the Histiocytosis Association.
unable to form foamy macrophages when exposed to strongly
macrophage-promoting conditions of M-CSF and lipid-containing
human serum. Both classical and nonclassical monocytes rapidly
differentiated into typical foam cells under these conditions. CD161 Authorship
nonclassical monocytes are likely to be heterogeneous and have also
been characterized as DC-like.38,39 It is possible that a subpopulation Contribution: P.M. designed research, performed experiments,
formed the macrophages observed in culture as the final density was analyzed data, and wrote the manuscript: V.B. designed research,
lower than for equivalent numbers of CD141 monocytes. Given the analyzed data, and contributed clinical data and patient material;
high proportion of BRAF-mutated alleles in CD161 monocytes, they C.M.B. designed research, performed experiments, and analyzed data;
may contribute to histiocytes in the lesions of ECD, although they are A.N. contributed clinical data and patient material; N.M. performed
reported to be unable to form giant cells. The occurrence of mutated experiments and analyzed data; S.B. contributed clinical data and
alleles in monocytes also presents the possibility that macrophages in patient material; M.H. designed research and contributed clinical
LCH lesions arise from the neoplastic clone. Previous studies show data and patient material; E.L.D., B.H.D., J.V., D.H., H.G., and
that M-CSF, one of the factors used to differentiate foam cells, is M.M. contributed clinical data and patient material; K.L.M. and C.A.
significantly increased in MS-LCH.36 designed research; O.A.-W. designed research and contributed clinical
An important caveat of the in vitro differentiation experiment is that data and patient material; and M.C. supervised the study, designed
healthy control cells without BRAF mutation were used. In the absence research, analyzed data, and wrote the manuscript.
of a surface antigen that can distinguish mutated and unmutated Conflict-of-interest disclosure: The authors declare no competing
progeny, this analysis cannot be performed with enriched BRAF- financial interests.
mutated cells isolated from patients. The presence of mutation may Correspondence: Matthew Collin, Human Dendritic Cell
not matter because ERK activation is a consequence of many of the Laboratory, Institute of Cellular Medicine, Newcastle University,
stimuli used in vitro. However, it remains hypothetically possible Framlington Pl, Newcastle upon Tyne NE2 4HH, United Kingdom;
that intrinsic BRAF activation has other unanticipated effects. e-mail: matthew.collin@ncl.ac.uk.

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